Targeting Epigenetic Mechanisms to Treat Alcohol Use Disorders (AUD).

BACKGROUND: The impact of abusive alcohol consumption on human health is remarkable. According to the World Health Organization (WHO), approximately 3.3 million people die annually because of harmful alcohol consumption (the figure represents around 5.9% of global deaths). Alcohol Use Disorder (AUD) is a chronic disease where individuals exhibit compulsive alcohol drinking and present negative emotional states when they do not drink. In the most severe manifestations of AUD, the individuals lose control over intake despite a decided will to stop drinking. Given the multiple faces and the specific forms of this disease, the term AUD often appears in the plural (AUDs). Since only a few approved pharmacological treatments are available to treat AUD and they do not apply to all individuals or AUD forms, the search for compounds that may help to eliminate the burden of the disease and complement other therapeutical approaches is necessary. METHODS: This work reviews recent research focused on the involvement of epigenetic mechanisms in the pathophysiology of AUD. Excessive drinking leads to chronic and compulsive consumption that eventually damages the organism. The central nervous system is a key target and is the focus of this study. The search for the genetic and epigenetic mechanisms behind the intricated dysregulation induced by ethanol will aid researchers in establishing new therapy approaches. CONCLUSION: Recent findings in the field of epigenetics are essential and offer new windows for observation and research. The study of small molecules that inhibit key epienzymes involved in nucleosome architecture dynamics is necessary in order to prove their action and specificity in the laboratory and to test their effectivity and safety in clinical trials with selected patients bearing defined alterations caused by ethanol.

Dexamethasone protects retinal ganglion cells but not Müller glia against hyperglycemia in vitro.

Diabetic retinopathy (DR) is a common complication of diabetes, for which hyperglycemia is a major etiological factor. It is known that retinal glia (Müller cells) and retinal ganglion cells (RGCs) are affected by diabetes, and there is evidence that DR is associated with neural degeneration. Dexamethasone is a glucocorticoid used to treat many inflammatory and autoimmune conditions, including several eye diseases like DR. Thus, our goal was to study the effect of dexamethasone on the survival of RGCs and Müller glial cells isolated from rat retinas and maintained in vitro under hyperglycemic conditions. The behavior of primary RGC cell cultures, and of mixed RGC and Müller cell co-cultures, was studied in hyperglycemic conditions (30 mM glucose), both in the presence and absence of Dexamethasone (1 µM). RGC and Müller cell survival was evaluated, and the conditioned media of these cultures was collected to quantify the inflammatory cytokines secreted by these cells using a multiplex assay. The role of IL-1ß, IL-6 and TNFα in RGC death was also evaluated by adding these cytokines to the co-cultures. RGC survival decreased significantly when these cells were grown in high glucose conditions, reaching 54% survival when they were grown alone and only 33% when co-cultured with Müller glia. The analysis of the cytokines in the conditioned media revealed an increase in IL-1ß, IL-6 and TNFα under hyperglycemic conditions, which reverted to the basal concentration in co-cultures maintained in the presence of dexamethasone. Finally, when these cytokines were added to co-cultures they appeared to have a direct effect on RGC survival. Hence, these cytokines could be implicated in the death of RGCs when glucose concentrations increase and dexamethasone might protect RGCs from the cell death induced in these conditions.

Glia-neuron interactions in the mammalian retina.

The mammalian retina provides an excellent opportunity to study glia-neuron interactions and the interactions of glia with blood vessels. Three main types of glial cells are found in the mammalian retina that serve to maintain retinal homeostasis: astrocytes, Müller cells and resident microglia. Müller cells, astrocytes and microglia not only provide structural support but they are also involved in metabolism, the phagocytosis of neuronal debris, the release of certain transmitters and trophic factors and K(+) uptake. Astrocytes are mostly located in the nerve fibre layer and they accompany the blood vessels in the inner nuclear layer. Indeed, like Müller cells, astrocytic processes cover the blood vessels forming the retinal blood barrier and they fulfil a significant role in ion homeostasis. Among other activities, microglia can be stimulated to fulfil a macrophage function, as well as to interact with other glial cells and neurons by secreting growth factors. This review summarizes the main functional relationships between retinal glial cells and neurons, presenting a general picture of the retina recently modified based on experimental observations. The preferential involvement of the distinct glia cells in terms of the activity in the retina is discussed, for example, while Müller cells may serve as progenitors of retinal neurons, astrocytes and microglia are responsible for synaptic pruning. Since different types of glia participate together in certain activities in the retina, it is imperative to explore the order of redundancy and to explore the heterogeneity among these cells. Recent studies revealed the association of glia cell heterogeneity with specific functions. Finally, the neuroprotective effects of glia on photoreceptors and ganglion cells under normal and adverse conditions will also be explored.

In vitro evaluation of the antielastase activity of polycyclic ß-lactams.

A series of bi- and tricyclic ß-lactam compounds was synthesized and evaluated as inhibitors of cleavage of synthetic substrates in vitro by the serine proteases Human Leukocyte Elastase (HLE), Human Leukocyte Proteinase 3 (HLPR3) and Porcine Pancreatic Elastase (PPE). The obtained results have permitted us to describe a homobenzocarbacephem compound as HLE and HLPR3 inhibitor, to observe the positive effect that the styryl group exerts on the HLE inhibitory activity in polycyclic ß-lactam compounds and to conclude that the hydroxyl function decreases the HLE inhibitory activity or rules it out completely.

Stem cell plasticity, neuroprotection and regeneration in human eye diseases.

Regeneration and plasticity refer to the ability of certain progenitor cells to produce cell lineages with specific morphological and functional settings. The pathway from a less delineated or immature phenotype to a mature or specialized one follows intricate routes where a monumental array of molecular elements, basically transcription factors and epigenetic regulators that turn off or on a specific phenotypic change, play a fundamental role. Nature itself offers procedures to healing strategies. Therapy approaches to pathologies in the realm of ophthalmology may benefit from the knowledge of the properties and mechanisms of activation of different routes controlling the pathways of cell definition and differentiation. Specification of cell identity, not only in terms of phenotypic traits, but also regarding the mechanisms of gene expression and epigenetic regulation, will provide new tools to manipulating cell fates and status, both forward and backwards. In the human eye, two main locations shelter stem cells: the limbus, which is situated in the limit of the cornea and the conjunctiva, and the ciliary body pars plana. Transplantation of limbal cells is currently used in certain pathologies where corneal epithelium is damaged. Therapeutic applications of retina progenitors are not yet fully developed due to the complexity of the cellular components of the multilayer retinal architecture. Animal models of Retinitis pigmentosa or Glaucoma offer an interesting approach to validate certain techniques, such as the direct injection of progenitors into the vitreal compartment, aimed to restoring retinal function.

Altered expression of retinal molecular markers in the canine RPE65 model of Leber congenital amaurosis.

PURPOSE: Leber congenital amaurosis (LCA) is a group of childhood-onset retinal diseases characterized by severe visual impairment or blindness. One form is caused by mutations in the RPE65 gene, which encodes the retinal pigment epithelium (RPE) isomerase. In this study, the retinal structure and expression of molecular markers for different retinal cell types were characterized, and differences between control and RPE65 mutant dogs during the temporal evolution of the disease were analyzed. METHODS: Retinas from normal and mutant dogs of different ages were examined by immunofluorescence with a panel of 16 different antibodies. RESULTS: Cones and rods were preserved in the mutant retinas, and the number of cones was normal. However, there was altered expression of cone arrestin and delocalization of rod opsin. The ON bipolar cells showed sprouting of the dendritic arbors toward the outer nuclear layer (ONL) and retraction of their axons in the inner nuclear layer (INL). A decreased expression of GABA, and an increased expression of intermediate filament glial markers was also found in the mutant retinas. These changes were more evident in the adult than the young mutant retinas. CONCLUSIONS: The structure of the retina is well preserved in the mutant retina, but several molecular changes take place in photoreceptors and in bipolar and amacrine cells. Some of these changes are structural, whereas others reflect a change in localization of the examined proteins. This study provides new information that can be applied to the interpretation of outcomes of retinal gene therapy in animal models and humans.

Immunohistochemical changes in rat retinas at various time periods of elevated intraocular pressure.

PURPOSE: To study alterations in different retinal cell types associated with retinal ganglion cell (RGC) death after elevation of intraocular pressure (IOP) in rats. METHODS: IOP was elevated by episcleral vein cauterization of the rat left eye. The right unoperated eye was kept as the control. IOP was measured when rats were awake. The animals were euthanized after one week (n=4) and five weeks (n=4). Their eyes were enucleated, postfixed, cryoprotected, and embedded in optimal cutting temperature (OCT) medium. Cryosections of the retina were cut at 14 microm thickness and processed for immunocytochemistry with 15 antibodies that specifically stain different retinal cell types. The distribution and intensity of the label was analyzed by comparing sections of control and glaucomatous retinas obtained from identical locations. RESULTS: The amount of amacrine cells identified by calcium binding proteins and choline acetyltransferase antibodies decreased after five weeks of elevated IOP. By using the anti-protein kinase C-alpha antibody, we were able to label a subpopulation of rod bipolar cells in control retinas but not in retinas that had elevated IOP. No changes were found in RGCs labeled with brain derived neurotrophic factor when comparing control and glaucomatous retinas. Glial fibrillary acidic protein and vimentin expression in glial cells increased after one week of elevated IOP. CONCLUSIONS: After one week of elevated IOP and before the onset of RGC death, it was evident that inner retinal cells showed remarkable changes in their molecular expression.