Molecular pathways in experimental glaucoma models.

Glaucoma is a complex and progressive disease that primarily affects the optic nerve axons, leading to irreversible vision loss. Although the exact molecular mechanisms underlying glaucoma pathogenesis are not fully understood, it is believed that except increased intraocular pressure, a combination of genetic and environmental factors play a role in the development of the disease. Animal models have been widely used in the study of glaucoma, allowing researchers to better understand the underlying mechanisms of the disease and test potential treatments. Several molecular pathways have been implicated in the pathogenesis of glaucoma, including oxidative stress, inflammation, and excitotoxic-induced neurodegeneration. This review summarizes the most important knowledge about molecular mechanisms involved in the glaucoma development. Although much research has been done to better understand the molecular mechanisms underlying this disease, there is still much to be learned to develop effective treatments and prevent vision loss in those affected by glaucoma.

Aland Island Eye Disease with Retinoschisis in the Clinical Spectrum of CACNA1F-Associated Retinopathy-A Case Report.

Aland island eye disease (AIED), an incomplete form of X-linked congenital stationary night blindness (CSNB2A), and X-linked cone-rod dystrophy type 3 (CORDX3) display many overlapping clinical findings. They result from mutations in the CACNA1F gene encoding the α1F subunit of the Cav1.4 channel, which plays a key role in neurotransmission from rod and cone photoreceptors to bipolar cells. Case report: A 57-year-old Caucasian man who had suffered since his early childhood from nystagmus, nyctalopia, low visual acuity and high myopia in both eyes (OU) presented to expand the diagnostic process, because similar symptoms had occurred in his 2-month-old grandson. Additionally, the patient was diagnosed with protanomalous color vision deficiency, diffuse thinning, and moderate hypopigmentation of the retina. Optical coherence tomography of the macula revealed retinoschisis in the right eye and foveal hypoplasia in the left eye. Dark-adapted (DA) 3.0 flash full-field electroretinography (ffERG) amplitudes of a-waves were attenuated, and the amplitudes of b-waves were abolished, which resulted in a negative pattern of the ERG. Moreover, the light-adapted 3.0 and 3.0 flicker ffERG as well as the DA 0.01 ffERG were consistent with severely reduced responses OU. Genetic testing revealed a hemizygous form of a stop-gained mutation (c.4051C>T) in exon 35 of the CACNA1F gene. This pathogenic variant has so far been described in combination with a phenotype corresponding to CSNB2A and CORDX3. This report contributes to expanding the knowledge of the clinical spectrum of CACNA1F-related disease. Wide variability and the overlapping clinical manifestations observed within AIED and its allelic disorders may not be explained solely by the consequences of different mutations on proteins. The lack of distinct genotype-phenotype correlations indicates the presence of additional, not yet identified, disease-modifying factors.

Glycosaminoglycans and collagen fibril distribution at various depths of the corneal stroma of normal and CXL treated rats.

Corneal collagen cross-linking (CXL) is widely used to treat keratoconus and ecstatic corneal disorders. The present studies were carried out to investigate the distribution of glycosaminoglycans (GAGs) and collagen fibril (CF) at different depths of the normal and CXL treated corneal stroma of four week old rats 7 days after standard CXL application. Ten Wistar rats' corneas were used for the study. The epithelium of the cornea from the left eye of each rat was removed and treated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The cornea from the right eye was used as the control cornea. The cornea was removed from the eye and processed for transmission electron microscopy. A bottom mounted Quemesa camera was used to capture digital images and these images were analysed using iTEM software. In the control cornea, the GAGs area size was not significantly different in the anterior, middle, and posterior stroma. In the CXL treated rats the GAGs area size gradually increased from the anterior to the posterior stroma whereas the spacing between the GAGs gradually decreased. There were very large GAGs present in the posterior stroma of the CXL treated rats. When comparing the control and CXL cornea, the GAGs area in the CXL cornea was significantly higher and inter-GAGs-spacing was smaller than in the control cornea. In the control cornea, the collagen fibrils diameter was higher in the anterior stroma and lowest in the posterior stroma. In the CXL treated cornea, the CF diameter and the interfibrillar spacing gradually decreased from the anterior to the posterior stroma. On comparison between the control and the CXL treated cornea, the interfibrillar spacing was significantly smaller in the CXL treated cornea than the control cornea in the anterior, middle, and posterior stroma but there was no difference in the diameter. The CXL treatment significantly increased the GAGs area and decreased the inter-GAGs-spacing, and inter-CF-spacing. This could be due to the gradual decline in the availability of riboflavin, UVA, and oxygen in the middle and posterior stroma. Further studies are required to investigate the role of keratan sulphate and chondroitin sulphate by using monoclonal antibodies with immunogold technique.

Extracellular vesicles in degenerative retinal diseases: A new therapeutic paradigm.

Nanoscale extracellular vesicles (EVs), consisting of exomers, exosomes and microvesicles/ectosomes, have been extensively investigated in the last 20 years, although their biological role is still something of a mystery. EVs are involved in the transfer of lipids, nucleic acids and proteins from donor to recipient cells or distant organs as well as regulating cell-cell communication and signaling. Thus, EVs are important in intercellular communication and this is not limited to sister cells, but may also mediate the crosstalk between different cell types even over long distances. EVs play crucial functions in both cellular homeostasis and the pathogenesis of diseases, and since their contents reflect the status of the donor cell, they represent an additional valuable source of information for characterizing complex biological processes. Recent advances in isolation and analytical methods have led to substantial improvements in both characterizing and engineering EVs, leading to their use either as novel biomarkers for disease diagnosis/prognosis or even as novel therapies. Due to their capacity to carry biomolecules, various EV-based therapeutic applications have been devised for several pathological conditions, including eye diseases. In the eye, EVs have been detected in the retina, aqueous humor, vitreous body and also in tears. Experiences with other forms of intraocular drug applications have opened new ways to use EVs in the treatment of retinal diseases. We here provide a comprehensive summary of the main in vitro, in vivo, and ex vivo literature-based studies on EVs' role in ocular physiological and pathological conditions. We have focused on age-related macular degeneration, diabetic retinopathy, glaucoma, which are common eye diseases leading to permanent blindness, if not treated properly. In addition, the putative use of EVs in retinitis pigmentosa and other retinopathies is discussed. Finally, we have reviewed the potential of EVs as therapeutic tools and/or biomarkers in the above-mentioned retinal disorders. Evidence emerging from experimental disease models and human material strongly suggests future diagnostic and/or therapeutic exploitation of these biological agents in various ocular disorders with a good possibility to improve the patient's quality of life.

The effectiveness and safety of one-stage iStent-based micro-invasive glaucoma surgery-A retrospective study.

Purpose: Micro-invasive glaucoma surgery involves a group of treatment methods associated with a low rate of side effects and good effectiveness outcomes. One of the most frequently performed procedures belonging to this group is iStent microstent implantation. The aim of this study was to perform a retrospective evaluation of the safety and efficacy of a combined procedure involving cataract phacoemulsification and single iStent microstent implantation, performed simultaneously. Materials and methods: The complete medical records of 62 patients (91 eyes) were analyzed retrospectively, including the best corrected visual acuity, intraocular pressure, the mean defect of visual fields, and the number of active substances used in eye drops. The follow-up times were 1, 3, 6, 9, and 12 months after the surgical procedure. Results: A significant improvement in the best corrected visual acuity and a reduction of the intraocular pressure were achieved after the surgery. On average, after 12 months, the best corrected visual acuity improved from 0.70 (0.25) to 0.91 (0.18; p = 0.001), the intraocular pressure reduced from 17.76 (3.95) to 14.91 (3.04; p = 0.0001), and the number of active substances used in eye drops reduced from 2.07 (1.08) to 0.70 (0.06; p = 0001). In addition, we found that patients who initially showed higher intraocular pressure values did not benefit from surgery in the aspect of the number of active substances used in their eye drops. Intraoperative and postoperative adverse events were transient and ultimately did not affect the outcomes. Conclusion: Simultaneous cataract phacoemulsification with single iStent implantation in patients with open-angle glaucoma is a safe and effective method for reducing intraocular pressure and the number of topical medications that must be used. Having initially higher intraocular pressure values may limit the beneficial effects of iStent implantation by subordinating patients from topical treatment; thus, single iStent implantation may not be the most favorable choice in uncontrolled glaucoma cases.

Metallothioneins, a Part of the Retinal Endogenous Protective System in Various Ocular Diseases.

Metallothioneins are the metal-rich proteins that play important roles in metal homeostasis and detoxification. Moreover, these proteins protect cells against oxidative stress, inhibit proapoptotic mechanisms and enhance cell differentiation and survival. Furthermore, MTs, mainly MT-1/2 and MT-3, play a vital role in protecting the neuronal retinal cells in the eye. Expression disorders of these proteins may be responsible for the development of various age-related eye diseases, including glaucoma, age-related macular degeneration, diabetic retinopathy and retinitis pigmentosa. In this review, we focused on the literature reports suggesting that these proteins may be a key component of the endogenous protection system of the retinal neurons, and, when the expression of MTs is disrupted, this system becomes inefficient. Moreover, we described the location of different MT isoforms in ocular tissues. Then we discussed the changes in MT subtypes' expression in the context of the common eye diseases. Finally, we highlighted the possibility of the use of MTs as biomarkers for cancer diagnosis.

Deficiency of the RNA-binding protein ELAVL1/HuR leads to the failure of endogenous and exogenous neuroprotection of retinal ganglion cells.

Introduction: ELAVL1/HuR is a keystone regulator of gene expression at the posttranscriptional level, including stress response and homeostasis maintenance. The aim of this study was to evaluate the impact of hur silencing on the age-related degeneration of retinal ganglion cells (RGC), which potentially describes the efficiency of endogenous neuroprotection mechanisms, as well as to assess the exogenous neuroprotection capacity of hur-silenced RGC in the rat glaucoma model. Methods: The study consisted of in vitro and in vivo approaches. In vitro, we used rat B-35 cells to investigate, whether AAV-shRNA-HuR delivery affects survival and oxidative stress markers under temperature and excitotoxic insults. In vivo approach consisted of two different settings. In first one, 35 eight-week-old rats received intravitreal injection of AAV-shRNA-HuR or AAV-shRNA scramble control. Animals underwent electroretinography tests and were sacrificed 2, 4 or 6 months after injection. Retinas and optic nerves were collected and processed for immunostainings, electron microscopy and stereology. For the second approach, animals received similar gene constructs. To induce chronic glaucoma, 8 weeks after AAV injection, unilateral episcleral vein cauterization was performed. Animals from each group received intravitreal injection of metallothionein II. Animals underwent electroretinography tests and were sacrificed 8 weeks later. Retinas and optic nerves were collected and processed for immunostainings, electron microscopy and stereology. Results: Silencing of hur induced apoptosis and increased oxidative stress markers in B-35 cells. Additionally, shRNA treatment impaired the cellular stress response to temperature and excitotoxic insults. In vivo, RGC count was decreased by 39% in shRNA-HuR group 6 months after injection, when compared to shRNA scramble control group. In neuroprotection study, the average loss of RGCs was 35% in animals with glaucoma treated with metallothionein and shRNA-HuR and 11.4% in animals with glaucoma treated with metallothionein and the scramble control shRNA. An alteration in HuR cellular content resulted in diminished photopic negative responses in the electroretinogram. Conclusions: Based on our findings, we conclude that HuR is essential for the survival and efficient neuroprotection of RGC and that the induced alteration in HuR content accelerates both the age-related and glaucoma-induced decline in RGC number and function, further confirming HuR's key role in maintaining cell homeostasis and its possible involvement in the pathogenesis of glaucoma.

Rodent Models of Diabetic Retinopathy as a Useful Research Tool to Study Neurovascular Cross-Talk.

Diabetes is a group of metabolic diseases leading to dysfunction of various organs, including ocular complications such as diabetic retinopathy (DR). Nowadays, DR treatments involve invasive options and are applied at the sight-threatening stages of DR. It is important to investigate noninvasive or pharmacological methods enabling the disease to be controlled at the early stage or to prevent ocular complications. Animal models are useful in DR laboratory practice, and this review is dedicated to them. The first part describes the characteristics of the most commonly used genetic rodent models in DR research. The second part focuses on the main chemically induced models. The authors pay particular attention to the streptozotocin model. Moreover, this section is enriched with practical aspects and contains the current protocols used in research in the last three years. Both parts include suggestions on which aspect of DR can be tested using a given model and the disadvantages of each model. Although animal models show huge variability, they are still an important and irreplaceable research tool. Note that the choice of a research model should be thoroughly considered and dependent on the aspect of the disease to be analyzed.

Surgical Menopause Impairs Retinal Conductivity and Worsens Prognosis in an Acute Model of Rat Optic Neuropathy.

Deficiency of estradiol during the menopausal period is an important risk factor for neurodegenerative diseases, including various optic neuropathies. The aim of this study was to evaluate the impact of surgical menopause on the function and survival ratio of RGCs in the rat model of ONC (optic nerve crush). We used eight-week-old female Long Evans rats, divided into two main groups depending on the time between ovariectomy procedure (OVA) and euthanasia (two weeks vs. seven weeks), and subgroups-OVA, OVA + ONC, or ONC. Retinal function was assessed with electroretinography (ERG). RGC loss ratio was evaluated using immunolabelling and counting of RGCs. Seven weeks after OVA, the menopause morphologically affected interneurons but not RGC; however, when the ONC procedure was applied, RGCs appeared to be more susceptible to damage in case of deprivation of estrogens. In our analysis, PhNR (photopic negative responses) were severely diminished in the OVA + ONC group. A deprivation of estrogens in menopause results in accelerated retinal neurodegeneration that firstly involves retinal interneurons. The lack of estrogens increases the susceptibility of RGCs to insults.

Nature-Inspired Hybrids (NIH) Improve Proteostasis by Activating Nrf2-Mediated Protective Pathways in Retinal Pigment Epithelial Cells.

Antioxidant systems play key roles in many elderly diseases, including age-related macular degeneration (AMD). Oxidative stress, autophagy impairment and inflammation are well-described in AMD, especially in retinal pigment epithelial (RPE) cells. The master regulator of antioxidant defense Nrf2 has been linked to AMD, autophagy and inflammation. In this study, in human ARPE-19 cells, some nature-inspired hybrids (NIH1-3) previously shown to induce Nrf2-mediated protection against oxidative stress were further investigated for their potential against cellular stress caused by dysfunction of protein homeostasis. NIH1-3 compounds increased the expression of two Nrf2-target genes coding defense proteins, HO-1 and SQSTM1/p62, in turn exerting beneficial effects on intracellular redox balance without modification of the autophagy flux. NIH1-3 treatments predisposed ARPE-19 cells to a better response to following exposure to proteasome and autophagy inhibitors, as revealed by the increase in cell survival and decreased secretion of the pro-inflammatory IL-8 compared to NIH-untreated cells. Interestingly, NIH4 compound, through an Nrf2-independent pathway, also increased cell viability and decreased IL-8 secretion, although to a lesser extent than NIH1-3, suggesting that all NIHs are worthy of further investigation into their cytoprotective properties. This study confirms Nrf2 as a valuable pharmacological target in contexts characterized by oxidative stress, such as AMD.

Effect of Ultraviolet-A and Riboflavin treatment on the architecture of the center and periphery of normal rat cornea: 7 days post treatment.

Corneal collagen cross-linking (CXL) is a treatment that is widely applied to halt the progression of ectatic diseases such as keratoconus by creating biomechanical strength in the cornea. Most of the studies assessed the effect of the CXL on the cornea without any differentiation of its effect between periphery and the center of the untreated control cornea especially after the 7 days of CXL application. We investigate the ultrastructural changes in the architecture of the center and periphery of rat corneas, 7 days after standard CXL application. Five Wistar rats (10 corneas) were used in the present study. The left eye corneas (5 mm area) were de-epithelialized and irradiated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The right eye corneas were used as a control. The sclera-cornea button was removed and processed for electron microscopy. Digital images were captured with a bottom mounted Quemesa camera and analyzed using the iTEM software. The ultrastructure of epithelium, hemi-desmosomes, Bowman's layer and stroma were organized in both untreated control and CXL rat cornea in both untreated control and CXL rat cornea. Within the same CXL cornea, both the collagen fibril (CF) diameter and interfibrillar spacing at the center were significantly smaller compared to the peripheral diameter and spacing of the cornea. When comparing the untreated control and CXL cornea, the central interfibrillar spacing of the CXL cornea was significantly smaller than the central spacing the untreated control cornea. In the CXL cornea the peripheral spacing was significantly higher compared to the peripheral interfibrillar spacing of the untreated control cornea. Within the CXL cornea, the proteoglycans (PGs) area and density of the periphery was significantly higher compared to the area and density of the center of the cornea. It suggests that CXL was more effective at the periphery of the cornea. This could be due to the higher amount of leucine rich PG lumican and higher diffusion of oxygen and riboflavin at the periphery cornea.

SCD1-Fatty Acid Desaturase Inhibitor MF-438 Alleviates Latent Inflammation Induced by Preservative-Free Prostaglandin Analog Eye Drops.

INTRODUCTION: Prostaglandin analogs are the first line of treatment in patients with glaucoma. Recently, many preservative-free prostaglandin analogs have been marketed to increase their tolerance in chronic use. However, potentially safer formulations have been reported to induce inflammation within ocular surface and adnexa, associated with pronounced activation of tissue macrophages. AIM: We aimed to evaluate the effect of a Stearoyl-CoA desaturase-1 (SCD1) inhibitor, MF-438, on the differentiation of monocytes exposed to eye drop detergents, representing saturated fatty acid derivatives. METHODS: A culture of human peripheral blood monocytes was exposed to eye drops containing fatty acid derivatives (eye drop detergents), pf-latanoprost (Monoprost®, hydroxystearate macrogolglycerol - MGHS40) or pf-tafluprost (Taflotan®, polysorbate 80 - PS80), as well as pf-latanoprost+MF-438, MGHS40, and PS80. For the negative control C(-), monocytes were cultured in basal medium, and for the positive controls, monocytes were stimulated with Lipopolysaccharide (LPS) and Interferon γ (IFNγ) (M1 macrophages) or Interleukin-4 (IL-4) (M2 macrophages). The concentration of desaturase in the cell homogenates was determined by ELISA. The number of cells was counted under a microscope at 20x magnification. RESULTS: The following concentrations of SCD1 (ng/mL) were measured: 7.8±0.3 - pf-latanoprost group; 1.5±0.4 - pf-tafluprost group; 6.8±0.7 - MGHS40 group; 0.4±0.002 - PS80 group; 0.9±0.02 - pf-latanoprost+MF-438 group; 5.4±1.6 - C(-) control; 0.5±0.04 - M1 control; 2.2±0.13 - M2 control. The percentages of macrophages in culture were 33.6%, 17.6%, 33%, 0%, 13.5%, 18.6%, 36.3%, and 39.3% for the pf-latanoprost, pf-tafluprost, MGHS40, PS80, pf-latanoprost+MF-438, C(-), M1, and M2 cultures, respectively. There was a strong correlation between SCD1 concentration and macrophage count in the culture (r=0.8, p<0.05). CONCLUSION: Inhibition of SCD1 in monocytes prevents their transformation into macrophages after exposure to saturated fatty acid derivatives contained in eye drops, which may contribute to the limitation of latent inflammation within ocular adnexa and could possibly translate into better tolerability of the topical treatment.

A unique pre-endothelial layer at the posterior peripheral cornea: ultrastructural study.

This study was conducted to investigate the ultrastructure of a unique structures at the anterior side of the endothelium of the posterior peripheral cornea and compare their inner fibers to those of the limbus and sclera. The unique structures at the anterior side of endothelium was referred as a pre-endothelial (PENL) structures in the present manuscript. Ten anonymous-donor human corneoscleral rims (leftover after corneal transplants) were processed for electron microscopy. Semi-thin sections were examined using an Olympus BX53 microscope, and ultrathin sections were studied using a JOEL 1400 transmission electron microscope. A unique PENL structures was identified at the posterior peripheral cornea at a radial distance of approximately 70-638 µm, from the endpoint of Descemet's membrane. The PENL thinned out gradually and disappeared in the center. The contained an electron-dense sheath with periodic structures (narrow-spacing fibers), wide-spacing fibers, and numerous microfibrils. Typical elastic fibers were present in the sclera and limbus but were not observed in the PENL. This study revealed the existence of a new acellular PENL, containing unique fibrillar structures that were unseen in the corneal stroma. From the evidence describe in this paper we therefore suggest that PENL is a distinct morphological structure present at the corneal periphery.

Ultrastructural study of collagen fibrils, proteoglycans and lamellae of the cornea treated with iontophoresis - UVA cross-linking and hypotonic riboflavin solution.

To investigate the effects of iontophoresis-ultraviolet A (UVA) cross-linking (CXL) with hypotonic riboflavin solution on the ultrastructural changes in the lamellae, collagen fibrils (CFs), and proteoglycans (PGs) in the central and peripheral stroma of the human corneal buttons. The iontophoresis method was used for the trans-epithelial application of hypotonic riboflavin in ex vivo corneal culture for 5 min. The corneas were irradiated using three methods: Group 1 (G1) , a UVA irradiance of 3 mW/cm2 for 30 min; Group 2 (G2) , a UVA irradiance of 10 mW/cm2 for 9 min; Group 3 (G3) , without UVA irradiation. Three untreated corneas were used as controls ( G0 ). After the CXL procedure, the corneas were processed for electron microscopy. The CF diameter and PGs in each sample were analyzed using the iTEM program. The keratocyte organelles and stromal architecture in the peripheral cornea were better preserved than those in the central cornea. In G1 and G2, the mean CF diameter in the peripheral cornea was significantly higher than that in the central cornea. In G3, the CF diameter in the central cornea was significantly larger than that in the peripheral cornea. Furthermore, differences in PG area size were observed between the central and peripheral corneas in all groups. Riboflavin + UVA application at 3 mW/cm2 for 30 min and 10 mW/cm2 for 9 min was a suitable method of CXL; however, 3 mW/cm2 for 30 min improved the organization and size of the collagen fibrils. CXL treatment applied at the periphery was more effective than that applied at the center.

Electrical synapses interconnecting axons revealed in the optic nerve head - a novel model of gap junctions' involvement in optic nerve function.

PURPOSE: To characterize newly discovered electrical synapses, formed by connexin (Cx) 36 and 45, between neighbouring axons within the optic nerve head. METHODS: Twenty-five Wistar rats were killed by CO2 inhalation. Proximal and distal optic nerve (ON) stumps were collected and processed for immunostainings, electron microscopy (EM) with immunogold labelling, PCR and Western blots (WB). Additional 15 animals were deeply anaesthetized, and flash visual evoked potentials (fVEP) after retrobulbar injection of saline (negative control) or 100 µm meclofenamic acid solution (gap junctions' blocker) were recorded. Human paraffin cross-sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research. RESULTS: Immunostainings of both rat and human ON revealed the presence of Cx45 and 36 colocalizing with ß3-tubulin, but not with glial fibrillary acidic protein (GFAP). In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 ± 0.005 in proximal stump and 0.44 ± 0.02 in distal stump), Cx45 showed higher levels (0.68 ± 0.01 in proximal stump and 0.9 ± 0.07 in distal stump). In immunogold-EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) directly coupling neighbouring axons. In fVEP, blocking of gap junctions with meclofenamic acid resulted in significant prolongation of the latency of P1 wave up to 160% after 30 min (p < 0.001). CONCLUSIONS: Optic nerve (ON) axons are equipped with electrical synapses composed of neuronal connexins, especially Cx45, creating direct morphological and functional connections between each other. This finding could have substantial implications for understanding of the pathogenesis of various optic neuropathies and identifies a new potential target for a therapeutic approach.

Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration.

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1α in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1α dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1α dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.

The Role of Endogenous Neuroprotective Mechanisms in the Prevention of Retinal Ganglion Cells Degeneration.

Retinal neurons are not able to undergo spontaneous regeneration in response to damage. A variety of stressors, i.e., UV radiation, high temperature, ischemia, allergens, and others, induce reactive oxygen species production, resulting in consecutive alteration of stress-response gene expression and finally can lead to cell apoptosis. Neurons have developed their own endogenous cellular protective systems. Some of them are preventing cell death and others are allowing functional recovery after injury. The high efficiency of these mechanisms is crucial for cell survival. In this review we focus on the contribution of the most recently studied endogenous neuroprotective factors involved in retinal ganglion cell (RGC) survival, among which, neurotrophic factors and their signaling pathways, processes regulating the redox status, and different pathways regulating cell death are the most important. Additionally, we summarize currently ongoing clinical trials for therapies for RGC degeneration and optic neuropathies, including glaucoma. Knowledge of the endogenous cellular protective mechanisms may help in the development of effective therapies and potential novel therapeutic targets in order to achieve progress in the treatment of retinal and optic nerve diseases.

Autophagy Stimulus Promotes Early HuR Protein Activation and p62/SQSTM1 Protein Synthesis in ARPE-19 Cells by Triggering Erk1/2, p38MAPK, and JNK Kinase Pathways.

RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD). HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal inhibitor MG132, HuR positively affects at posttranscriptional level p62 expression, a stress response gene involved in protein aggregate clearance with a role in AMD. Here, we studied the early effects of the proautophagy AICAR + MG132 cotreatment on the HuR-p62 pathway. We treated ARPE-19 cells with Erk1/2, AMPK, p38MAPK, PKC, and JNK kinase inhibitors in the presence of AICAR + MG132 and evaluated HuR localization/phosphorylation and p62 expression. Two-hour AICAR + MG132 induces both HuR cytoplasmic translocation and threonine phosphorylation via the Erk1/2 pathway. In these conditions, p62 mRNA is loaded on polysomes and its translation in de novo protein is favored. Additionally, for the first time, we report that JNK can phosphorylate HuR, however, without modulating its localization. Our study supports HuR's role as an upstream regulator of p62 expression in ARPE-19 cells, helps to understand better the early events in response to a proautophagy stimulus, and suggests that modulation of the autophagy-regulating kinases as potential therapeutic targets for AMD may be relevant.

FluoroGold-Labeled Organotypic Retinal Explant Culture for Neurotoxicity Screening Studies.

Preclinical toxicity screening of the new retinal compounds is an absolute requirement in the pathway of further drug development. Since retinal neuron cultivation and in vivo studies are relatively expensive and time consuming, we aimed to create a fast and reproducible ex vivo system for retinal toxicity screening. For this purpose, we used rat retinal explant culture that was retrogradely labeled with the FluoroGold before the isolation. Explants were exposed to a toxic concentration of gentamicin and ciliary neurotrophic factor (CNTF), a known neuroprotective agent. The measured outcomes showed the cell density in retinal ganglion cell layer (GCL) and the activity of lactate dehydrogenase (LDH) in the culture medium. Gentamicin-induced oxidative stress resulted in retinal cell damage and rapid LDH release to the culture medium (p < 0.05). Additional CNTF supplementation minimized the cell damage, and the increase of LDH release was insignificant when compared to LDH levels before gentamicin insult (p > 0.05). As well as this, the LDH activity was directly correlated with the cell count in GCL (R = -0.84, p < 0.00001), making a sensitive marker of retinal neuron damage. The FLOREC protocol could be considered as a fast, reproducible, and sensitive method to detect neurotoxicity in the screening studies of the retinal drugs.