F. David Rodríguez

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.

G proteins coupled to phospholipase C : molecular targets of long-term ethanol exposure

Abstract: Long‐term ethanol exposure is known to inhibit bradykinin‐stimulated phosphoinositide hydrolysis in cultures of neuroblastoma × glioma 108–15 cells. In the present study, [3H]bradykinin binding, GTP‐binding protein function, and phospholipase C activity were assayed in cells grown for 4 days in 100 mM ethanol with the aim of elucidating the molecular target of ethanol on signal transduction coupled to inositol trisphosphate and diacylglycerol formation. Ethanol exposure reduced guanosine 5′‐O‐(3‐thiotriphosphate) [GTP(S)]‐ and, to a lesser extent, NaF/AlCl3‐stimulated phosphoinositide hydrolysis, whereas it had no effect on the enzymatic activity of a phosphatidylinositol 4,5‐bisphosphate‐specific phospholipase C. [3H]Bradykinin binding in the absence of GTP(S) was not influenced by ethanol exposure. However, the reduction in [3H]bradykinin binding seen in control cells after addition of GTP analogue was inhibited in cells grown in ethanol‐containing medium. The results indicate that long‐term ethanol exposure exerts its effects on receptor‐stimulated phosphoinositide hydrolysis primarily at the level of the GTP‐binding protein.

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.

Ethanol-Induced Changes in Signal Transduction via Formation of Phosphatidylethanol

In 1984 we demonstrated that phosphatidylethanol (Peth) was formed in different organs of ethanol-treated rats (Alling et al., 1984). Peth is a unique, anionic phospholipid formed in cell membranes only in the presence of ethanol. The highest concentration of Peth was found in membrane-rich organs such as brain and kidney but it could also be detected in liver, skeletal muscle and heart (Alling et al., 1983; Alling et al., 1984; Benthin et al., 1985). In addition to rats intoxicated with ethanol, Peth was also formed in cultured cells exposed to ethanol (Lundqvist et al., 1993).

GEMSP exerts a myelin-protecting role in the rat optic nerve.

Abstract Objectives: Chronic experimental autoimmune encephalomyelitis (EAE) was induced in rats to evaluate the potential protective effect of GEMSP, a mixture made up of fatty acids (FA), vitamins, and amino acids or their derivatives, linked to Poly-L-Lysine, on the myelin sheath of the optic nerve. Methods: To evaluate the effects of GEMSP on the optic nerve, animals were divided into three experimental groups: (1) EAE rats treated with GEMSP; (2) EAE rats treated with 0·9% NaCl; and (3) control, non-EAE rats. Using electron microscopy, we investigated the possibility that this new drug candidate has a myelin-protective role. Results: A marginally significant reduction in the thickness of the myelin around optic nerve medium-size axons (diameter between 0·8–1·3 μm) was found in EAE rats. Treatment of EAE rats with GEMSP ameliorated myelin damage. Significantly increased myelin thickness was found when animals in groups 2 and 3 were compared. However, the number of myelinated axons studied was not altered in groups 1 or 2 when compared to controls. Discussion: Our results suggest that in a model of demyelination, GEMSP protects and enhances the formation of the myelin sheath of the optic nerve and therefore could be a potential drug candidate to reduce optic nerve pathogenesis in multiple sclerosis (MS).

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

A series of bi- and tricyclic β-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 β-lactam compounds and to conclude that the hydroxyl function decreases the HLE inhibitory activity or rules it out completely.

Phospholipase C Coupled G-Proteins: Molecular Targets of Ethanol

The transfer of information into the intracellular compartment is an important step in neurotranmission. GTP-binding proteins (G-proteins) mediate this signalling and it is by now well established that ethanol differentially interacts with this set of proteins (reviewed by Hoffman and Tabakoff, 1990, Hoek et al., 1992). Most of the knowledge generated is connected to signalling through the adenylate cyclase system. Less is known about the role of phospholipase C coupled G-proteins. In analogy with the effect of ethanol on the cAMP system, it can be postulated that long-term ethanol may affect receptor-stimulated phosphoinositide hydrolysis by acting on the G-proteins coupled to this system. It is known that this is indeed the case in systems that are sensitive to acute exposure to alcohols (Rubin and Hoek, 1988, Rooney et al., 1989).

Repair of corneal damage with stem cells

The essence of regeneration and plasticity lies in the capacities of certain cell populations to give rise to progenies with specific functional and morphological traits. An array of molecular events directs this process (for instance, activation and de-activation of transcription or regulation of epigenetic mechanisms and controls). The unravelling of the processes that activate differentiation or de-differentiation events and the isolation and precise characterization of specific stem cell populations will open new avenues of therapy intervention in all areas of regenerative medicine, including eye pathologies. In the human anterior segment of the eye, adult stem cells can be found in the corneal limbus (the rim that separates cornea and conjunctiva). Currently, different approaches use transplantation of limbal epithelial stem cells (LESC) or corneal stromal stem cells (CSSC) to restore damaged cornea. LESC and CSSC establish a molecular dialogue that may support the maintenance of their stem phenotype. To restore corneal transparency and function other therapy approaches include the use of adult stem cells of different origins, bioengineered cells and biomaterials. Introduction According to The World Health Organization (WHO), corneal blindness (5.1% of total cases of blindness or visual deterioration) represents the fourth cause of blindness globally, after cataract, glaucoma and age-related macular degeneration (AMD). Updated advances in the application of stem cells to treat diseased cornea are reviewed in this work. Also, plasticity, “stemness” and regeneration are considered in the field of therapy endeavours targeted to tackle corneal pathologies. Stem cells have an essential role in development, tissue replacement and tissue repair. They reside in niches where an orchestrated ensemble of autocrine, paracrine and endocrine factors regulate their function and fate [1-3]. Stem cells are able to proliferate and differentiate into different cell types. Hence they are very important in cell renewal, both naturally and as a therapy tool. Difficulties in effective treatments are sometimes due to the significant extent and gravity of the lesion produced by both external insults (such as pathogenic agents or accidental damage due to burn or chemical corrosion) and genetic abnormality or ill-function. The search for new and effective treatments to restore vision is therefore a paramount. It is in this context where cell therapy may have an important niche of action. To regenerate a tissue to its partial or complete functional state new cells with high transformation potential should be obtained. Therefore, regeneration is based on appropriate replacement. Cell can be reprogrammed to an undifferentiated state from a differentiated one [4,5]. Also, some cell populations may shift among different states of differentiation. Anuran amphibians, for example, are able to regenerate the retina by means of a transdifferentiation process of the retinal pigmented epithelium and obtain a new lens from dorsal iris pigmented epithelium [6]. Cell differentiation is an intricate route that may progress in different directions. The complexities recline in molecular “orders” that carve the final fully functional cell. But the process can, at certain points, be stopped or reversed in opposite direction, thus making the pathway more flexible and prone to required adaptations [4,7]. In general, the term “stemness” refers to the dormant state and the capacity that some cells have to differentiate in given conditions [8]. But the expression incorporates different transformation capacities (totipotent stem cells exhibit the potential to generate any cell of an organism; an embryonic stem cell, however, generates all the cells of a given organism, but the trophoblast, and the production of progenies by postnatal stem cells is restricted to the tissue where they dwell [8,9]. Common characteristics of stem cells are their ability to divide and maintain their division potential or differentiate and loose such capacity [10]. Cell division can be symmetrical, where two identical cells are generated. When cell division is asymmetrical, one daughter cell keeps “stemness” whereas the other differentiates [8,10]. How and when the cell “decides” between symmetrical and asymmetrical divisions is not fully known but both external and intrinsic factors are involved [10]. The molecular machinery (noteworthy, control and modulation of transcription) responsible for the capacity of a cell to maintain a given state of “stemness” reacts to different and numerous stimuli [11,12]. It is important therefore to define the molecular events that determine cell potencies and fates. Once we have the knowledge, and expertise, Correspondence to: F. David Rodriguez, Department of Biochemistry and Molecular Biology, Group BMD, Faculty of Chemical Sciences, University of Salamanca, E-37007 Salamanca, Spain, Tel: +34 923294698, Fax: +34 923294579, E-mail: lario@usal.es

The inhibition of [3H] inositol 1,4,5-trisphosphate binding by Ca2+ is modified after long-term ethanol treatment and ethanol withdrawal

We have analysed the influence of long‐term ethanol exposure on the effect exerted by Ca 2+ on the binding of tritiated inositol 1,4,5‐trisphosphate to its receptors in rat cerebellar membranes. After 21 days of ethanol treatment the binding of the agonist was reduced in the absence of Ca 2+. The decrease was due to reduction in B max without any alteration of K d. In membranes from control animals Ca 2+ inhibited the binding of InsP 3 in a dose‐dependent manner by altering the affinity of the protein for the ligand. However, the inhibitory effect of Ca 2+ was abolished following chronic ethanol exposure. Five days after withdrawing ethanol, the B max recovered to control values, but the inhibitory effect of Ca 2+ was recovered at only 10 days after withdrawal. The results indicate that long‐term ethanol exposure may have differential effects on the InsP 3 binding site and on the Ca 2+ binding site, or alternatively on a Ca 2+ ‐related regulatory cycle.