Pedro de la Villa

Cellular responses following retinal injuries and therapeutic approaches for neurodegenerative diseases.

Retinal neurodegenerative diseases like age-related macular degeneration, glaucoma, diabetic retinopathy and retinitis pigmentosa each have a different etiology and pathogenesis. However, at the cellular and molecular level, the response to retinal injury is similar in all of them, and results in morphological and functional impairment of retinal cells. This retinal degeneration may be triggered by gene defects, increased intraocular pressure, high levels of blood glucose, other types of stress or aging, but they all frequently induce a set of cell signals that lead to well-established and similar morphological and functional changes, including controlled cell death and retinal remodeling. Interestingly, an inflammatory response, oxidative stress and activation of apoptotic pathways are common features in all these diseases. Furthermore, it is important to note the relevant role of glial cells, including astrocytes, Müller cells and microglia, because their response to injury is decisive for maintaining the health of the retina or its degeneration. Several therapeutic approaches have been developed to preserve retinal function or restore eyesight in pathological conditions. In this context, neuroprotective compounds, gene therapy, cell transplantation or artificial devices should be applied at the appropriate stage of retinal degeneration to obtain successful results. This review provides an overview of the common and distinctive features of retinal neurodegenerative diseases, including the molecular, anatomical and functional changes caused by the cellular response to damage, in order to establish appropriate treatments for these pathologies.

The protein kinase DYRK1A regulates caspase-9-mediated apoptosis during retina development.

The precise regulation of programmed cell death is critical for the normal development of the nervous system. We show here that DYRK1A (minibrain), a protein kinase essential for normal growth, is a negative regulator of the intrinsic apoptotic pathway in the developing retina. We provide evidence that changes in Dyrk1A gene dosage in the mouse strongly alter the cellularity of inner retina layers and result in severe functional alterations. We show that DYRK1A does not affect the proliferation or specification of retina progenitor cells, but rather regulates the number of cells that die by apoptosis. We demonstrate that DYRK1A phosphorylates caspase-9 on threonine residue 125, and that this phosphorylation event is crucial to protect retina cells from apoptotic cell death. Our data suggest a model in which dysregulation of the apoptotic response in differentiating neurons participates in the neuropathology of diseases that display DYRK1A gene-dosage imbalance effects, such as Downs syndrome.

Balance between autophagic pathways preserves retinal homeostasis

Aging contributes to the appearance of several retinopathies and is the largest risk factor for aged‐related macular degeneration, major cause of blindness in the elderly population. Accumulation of undegraded material as lipofuscin represents a hallmark in many pathologies of the aged eye. Autophagy is a highly conserved intracellular degradative pathway that plays a critical role in the removal of damaged cell components to maintain the cellular homeostasis. A decrease in autophagic activity with age observed in many tissues has been proposed to contribute to the aggravation of age‐related diseases. However, the participation of different autophagic pathways to the retina physiopathology remains unknown. Here, we describe a marked reduction in macroautophagic activity in the retina with age, which coincides with an increase in chaperone‐mediated autophagy (CMA). This increase in CMA is also observed during retinal neurodegeneration in the Atg5flox/flox; nestin‐Cre mice, a mouse model with downregulation of macroautophagy in neuronal precursors. In contrast to other cell types, this autophagic cross talk in retinal cells is not bi‐directional and CMA inhibition renders cone photoreceptor very sensitive to stress. Temporal and cell‐type‐specific differences in the balance between autophagic pathways may be responsible for the specific pattern of visual loss that occurs with aging. Our results show for the first time a cross talk of different lysosomal proteolytic systems in the retina during normal aging and may help the development of new therapeutic intervention for age‐dependent retinal diseases.

Two types of calcium currents of the mouse bipolar cells recorded in the retinal slice preparation

In the vertebrate retina, the bipolar cell makes reciprocal synapses with amacrine cells at the axon terminal. It has been postulated that amacrine cells may control the transmitter release from bipolar cells by modulating their calcium currents (ICa). To clarify this possibility calcium currents were studied in bipolar cells of the mouse retina using a slice preparation. ICa was identified by voltage clamp protocols, ionic substitution and pharmacological tools.

Functional modifications in rod bipolar cells in a mouse model of retinitis pigmentosa

The rd mouse has been widely used as an animal model of retinitis pigmentosa. In this model, a mutation of rod-specific phosphodiesterase leads to a loss of rods during the early period of postnatal life. Morphological modifications at the level of the outer plexiform layer have been shown (Proc. Nat. Acad. Sci. USA 97 (2000) 11020) in bipolar and horizontal cells. However, very little is known about the functional changes suffered by these cells postsynaptic to the degenerated rods. In the present work we have studied the neurotransmitter-induced currents in rod bipolar cells from the rd mouse retina. Currents induced by glutamate and GABA were studied by the patch clamp-whole cell technique, on rod bipolar cells enzymatically dissociated from the rd mouse retina. Data from rd animals were compared with non-dystrophic NMRI mice. GABA (30-100 micro M) and glutamate (100 micro M) were applied from a puff pipette in the near proximity of rod bipolar cell dendrites, clamped at physiological membrane potentials, and their evoked currents were studied. In rod bipolar cells from non-dystrophic mouse, puff application of glutamate induced an outward current. This current was increased twofold in absence of extracellular calcium (nominally 0 calcium). In rod bipolar cells from adult rd mouse, currents induced by glutamate were absent. Two types of GABA mediated currents were isolated in rod bipolar cells both in control and rd mouse retinas. The currents mediated by GABA(C) receptors were observed exclusively at the axon terminal, while the currents mediated by the GABA(A) receptors were observed upon GABA application to the bipolar cell dendrites. The currents mediated by GABA(A) receptors in rod bipolar cells from rd mouse were larger than those from control animals. We conclude that after the degeneration of rod photoreceptors in rd mouse, rod bipolar cells lost their glutamate (rod-neurotransmitter) input while they increase their response to GABA (horizontal cell-neurotransmitter). In our opinion, this work describes for the first time the changes in neurotransmitter sensitivity that affect rod bipolar cells after photoreceptor degeneration of the mouse retina.

Changes in the inner and outer retinal layers after acute increase of the intraocular pressure in adult albino Swiss mice

In adult albino mice the effects of increased intraocular pressure on the outer retina and its circuitry was investigated at intervals ranging 3-14 weeks. Ocular hypertension (OHT) was induced by cauterizing the vessels draining the anterior part of the mice eye, as recently reported (Salinas-Navarro et al., 2009a). Electroretinographic (ERG) responses were recorded simultaneously from both eyes and compared each other prior to and at different survival intervals of 2, 8 or 12 weeks after lasering. Animals were processed at 3, 9 or 14 weeks after lasering, and radial sections were obtained in the cryostat and further processed for immunocytochemistry using antibodies against recoverin, gamma-transducin, Protein Kinase C-alpha (PKC-alpha), calbindin or synaptophysin. The synaptic ribbons were identified using an antibody against the protein bassoon, which labels photoreceptor ribbons and nuclei were identified using TO-PRO. Laser photocoagulation of the perilimbar and episcleral veins of the left eye resulted in an increase in mean intraocular pressure to approximately over twice its baseline by 24 h that was maintained for approximately five days reaching basal levels by 1 week. ERG recordings from the different groups of mice showed their a-, b-wave and scotopic threshold response (STR) amplitudes, when compared to their contralateral fellow eye, reduced to 62%, 52% and 23% at 12 weeks after lasering. Three weeks after lasering, immunostaining with recoverin and transducin antibodies could not document any changes in the outer nuclear layer (ONL) but both ON-rod bipolar and horizontal cells had lost their dendritic processes in the outer plexiform layer (OPL). Sprouting of horizontal and bipolar cell processes were observed into the ONL. Fourteen weeks after lasering, protein kinase-C antibodies showed morphologic changes of ON-rod bipolar cells and calbindin staining showed abnormal horizontal cells and a loss of their relationship with their presynaptic input. Moreover, at this time, quantitative studies indicate significant diminutions in the number of photoreceptor synaptic ribbons/100 microm, and in the thickness of the outer nuclear and plexiform layer, when compared to their fellow eyes. Increased intraocular pressure in Swiss mice results in permanent alterations of their full field ERG responses and in changes of the inner and outer retinal circuitries.

Ectopic expression of tyrosine hydroxylase in the pigmented epithelium rescues the retinal abnormalities and visual function common in albinos in the absence of melanin

Albino mammals have profound retinal abnormalities, including photoreceptor deficits and misrouted hemispheric pathways into the brain, demonstrating that melanin or its precursors are required for normal retinal development. Tyrosinase, the primary enzyme in melanin synthesis commonly mutated in albinism, oxidizes l‐tyrosine to l‐dopaquinone using l‐3,4‐dihydroxyphenylalanine (L‐DOPA) as an intermediate product. L‐DOPA is known to signal cell cycle exit during retinal development and plays an important role in the regulation of retinal development. Here, we have mimicked L‐DOPA production by ectopically expressing tyrosine hydroxylase in mouse albino retinal pigment epithelium cells. Tyrosine hydroxylase can only oxidize l‐tyrosine to L‐DOPA without further progression towards melanin. The resulting transgenic animals remain phenotypically albino, but their visual abnormalities are corrected, with normal photoreceptor numbers and hemispheric pathways and improved visual function, assessed by an increase of spatial acuity. Our results demonstrate definitively that only early melanin precursors, L‐DOPA or its metabolic derivatives, are vital in the appropriate development of mammalian retinae. They further highlight the value of substituting independent but biochemically related enzymes to overcome developmental abnormalities.

Proinsulin/insulin is synthesized locally and prevents caspase- and cathepsin-mediated cell death in the embryonic mouse retina

Programmed cell death is an essential, highly regulated process in neural development. Although the role of insulin‐like growth factor I in supporting the survival of neural cells has been well characterized, studies on proinsulin/insulin are scarce. Here, we characterize proinsulin/insulin effects on cell death in embryonic day 15.5 mouse retina. Both proinsulin mRNA and proinsulin/insulin immunoreactivity were found in the developing retina. Organotypic embryonic day 15.5 retinas cultured under growth factor deprivation showed an increase in cell death that was reversed by proinsulin, insulin and insulin‐like growth factor I, with similar median effective concentration values via phosphatidylinositol‐3‐kinase activation. Although insulin and insulin‐like growth factor I provoked a sustained Akt phosphorylation, proinsulin‐induced phosphorylation of Akt was not found. Analysis of the growth factor deprivation‐induced cell death mechanisms, using caspase and cathepsin inhibitors, demonstrated that both protease families were required for the effective execution of cell death. The insulin survival effect, which decreased the extent and distribution of cell death to levels similar to those found in vivo, was not enhanced by simultaneous treatment with caspase and cathepsin inhibitors, suggesting that insulin interferes with these protease pathways in the embryonic mouse retina. The mechanisms characterized in this study provide new details on early neural cell death and its genuine regulation by insulin/proinsulin.

Depolarizing effect of GABA in rod bipolar cells of the mouse retina

Gamma-amino butyric acid (GABA) has been characterized as inhibitory neurotransmitter through chloride mediated channels in the adult nervous system. However, using gramicidin perforated patch-clamp recordings from rod bipolar cells dissociated from retinas of adult mice, we find that GABA is capable of inducing cell depolarization. Currents mediated by GABA(A) and GABA(C) receptors were further isolated by the use of GABA receptor specific blockers. In rod bipolar cells dissociated from the mouse retina, activation of GABA(A) receptors located at the cell dendrites induces ionic currents which show a reversal potential of -33 mV. However, local activation of GABA(C) receptors located at the axon terminal induces ionic currents with a reversal potential of -60 mV. According to Nernst equation, the dendrites of rod bipolar cells of the mouse retina would have a high intracellular chloride concentration ([Cl(-)](i)) and there must be an intracellular gradient in [Cl(-)](i), being the [Cl(-)](i) more elevated in the dendrites than in the axon terminal. The depolarizing effect of GABA at the dendrites of rod bipolar cells may contribute to the lateral interaction in the mammalian retina, thereby enhancing visual discrimination of stimuli input.

The effects of GABA and glycine on horizontal cells of the rabbit retina

Intracellular and patch-clamp recordings have been used to characterize GABA-activated channels in axonless horizontal cells (ALHC) of the rabbit retina. In our intracellular recordings on an everted eyecup preparation, GABA depolarized the horizontal cells (HC), diminished their light response amplitude and slowed the response rise time. Glycine showed similar effects on the HC light responses. In our whole cell patch-clamp recordings on dissociated ALHC, all HCs responded to 3 microM GABA but none to glycine, even at 100 microM. Dose-response relationship for GABA gave EC50 values around 10 microM and Hill slopes of 1.3. Whole-cell current-voltage (I-V) relationships of GABA-activated currents reversed close to the predicted Cl- equilibrium potential. Partial replacement of intracellular Cl- with isothetionate shifted the GABA reversal potential to a more negative value. Muscimol (30 microM), a GABAA agonist mimicked the effect of GABA, but baclofen (30 microM), a GABAB agonist and cis-aminocaprionic acid (30 microM), a GABAC agonist did not elicit any effect on ALHC. Responses to GABA were blocked by the GABAA receptor antagonist bicuculline (10 microM) and picrotoxin (100 microM). According to our results, we conclude that ALHC express GABA receptors coupled to ion channels, and they correspond to GABAA receptor subtypes.