Challenges in the Development of Therapy for Dry Age-Related Macular Degeneration.

Dry age-related macular degeneration (AMD), a multifactorial progressive degenerative disease of the retinal photoreceptors, pigmented epithelium and Bruch's membrane/choroid in central retina, causes visual impairment in millions of elderly people worldwide. The only available therapy for this disease is the over-the-counter (OTC) multi-vitamins plus macular xanthophyll (lutein/zeaxanthin) which attempts to block the damages of oxidative stress and ionizing blue light. Therefore development of dry AMD prescribed treatment is a pressing unmet medical need. However, this effort is currently hindered by many challenges, including an incomplete understanding of the mechanism of pathogenesis that leads to uncertain targets, confounded by not yet validated preclinical models and the difficulty to deliver the drugs to the posterior segment of the eye. Additionally, with slow disease progression and a less than ideal endpoint measurement method, clinical trials are necessarily large, lengthy and expensive. Increased commitment to research and development is an essential foundation for dealing with these problems. Innovations in clinical trials with novel endpoints, nontraditional study designs and the use of surrogate diseases might shorten the study time, reduce the patient sample size and consequently lower the budget for the development of the new therapies for the dry AMD.

Modulation of synaptic function by cGMP and cGMP-gated cation channels.

Cyclic nucleotide-gated cation channels have been studied intensively in the primary sensory neurons of the visual and olfactory systems. Using both anatomical and physiological methods we have shown that they have a much more widespread distribution in the nervous system. In many retinal ganglion cells cGMP, but not cAMP, activates a non-selective conductance that has many of the properties of CNG channels. As many neurons also contain cGMP-dependent protein kinases (PKGs), we have used a variety of cGMP analogues to distinguish the actions of cGMP. Sp-8-Br-PET-cGMPS is a potent non-hydrolyzable cGMP analogue that is an agonist of PKG. We found that Sp-8-Br-PET-cGMPS acts as a competitive inhibitor of at least the rod CNG channel. Rp-8-Br-cGMPS has shown the opposite effects, namely as an agonist of the rod CNG channel and an inhibitor of PKG. In dissociated cell cultures and slices of rodent visual cortex cGMP had multiple rapid and reversible effects on transmission at glutamatergic synapses. Extracellular application of 8-Br-cGMP or Sp-8-Br-PET-cGMPS reduced stimulus evoked EPSPs in cortical slices. In cortical cultures both analogs reduced the frequency of spontaneous EPSCs, but not their amplitude. The effects on both EPSPs and EPSCs were presynaptic. The effects on evoked EPSPs may be due, in part, to reduced calcium influx through voltage-gated calcium channels. The effects on spontaneous EPSCs may be due, in part, to modulation of calcium fluxes through internal stores. Similar modulations of synaptic transmission have been found at gabaergic synapses. On postsynaptic cells, PKG activation produced a dramatic enhancement of the responses to applied NMDA. No effects were detected on applied AMPA/kainate or GABA. Together the results suggest that cGMP may use multiple mechanisms to modulate synaptic efficacy and that its actions may include regulating synaptic plasticity and the relative strength of excitatory and inhibitory drive through neural pathways.

Expression and activation of STAT proteins during mouse retina development.

Cytokines and growth factors play important roles in mammalian ocular development and maintenance. Recent studies have indicated that some of these ligands can activate signal transducer and activator of transcription factors (STATs) and modulate gene transcription. The purpose of this study was to investigate the expression and activation of STAT proteins in the developing mouse retina. Anti-STAT and anti-phosphorylated STAT antibodies were used to detect the expression and activation of STATs in embryonic and postnatal neuronal retina, ciliary margin, and retinal pigment epithelium (RPE). In situ hybridization and Western blot were also employed. In embryonic stages, all STAT proteins were expressed in the neuronal retina in distinct cell populations at different embryonic stages. For example, Stat3 expression and activation gradually increased in the inner neuroblast layer and ciliary margin during development. In adult retina, Stat3 was detected in the inner nuclear layer and ganglion cells layers. Stat1 was strongly expressed in both outer and inner plexiform layers. Stat5a was clearly expressed in the outer/inner nuclear layer, the ganglion cell layer, and the inner plexiform layer. Strong expression of Stat3, Stat5a, and Stat6 was observed in the RPE. Activated Stat3 and Stat5a were found in the neural retina and the RPE. Distinct STAT proteins were present in different cell populations in neuronal retina and RPE suggesting multiple functions of STATs in mammalian eye development. Studies of STAT signal pathways in the eye may contribute to the understanding of molecular mechanisms in control of ocular development and pathogenesis.

Protective effect of arachidonic acid on glutamate neurotoxicity in rat retinal ganglion cells.

PURPOSE: Low concentrations of excitotoxic agents such as glutamate decrease survival of retinal ganglion cells (RGCs) and may be an important cause of RGC death in a variety of retinal diseases. Arachidonic acid (AA), an intercellular messenger in the central nervous system, has been reported to have multiple effects on glutamate receptors, including an inhibitory effect on non-N-methyl-D-aspartate (NMDA) receptors. The purpose of this study was to test the hypothesis that AA could protect RGCs from glutamate neurotoxicity. METHODS: RGCs were purified from the rat retina on postnatal days 7 and 8 by a modified two-step panning method. Survival of RGCs after exposure to glutamate, with or without AA treatment, was measured after 3 days in culture. To visualize calcium signals, RGCs were loaded with a calcium indicator dye, fluo-3 acetoxymethyl ester, and the fluorescence was measured by laser scanning confocal microscopy. Electrophysiological effects of AA on non-NMDA ionotropic receptors were examined by using whole-cell patch clamp configurations. RESULTS: Incubation of RGCs with 25 microM glutamate caused 60% loss of RGCs. This glutamate neurotoxicity was significantly ameliorated by low concentrations of AA. Concentrations of AA above 10 microM were toxic to RGCs. Calcium imaging showed that glutamate-, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- (AMPA) and kainate-induced intracellular calcium accumulation in these cells was reduced by AA. Electrophysiological recordings revealed that currents mediated by non-NMDA ionotropic receptors were inhibited by AA in a dose-dependent manner. CONCLUSIONS: Low concentrations of AA can reduce glutamate neurotoxicity to RGCs by the inhibition of non-NMDA ionotropic receptors. These results suggest that endogenous or exogenous AA may be used to protect RGCs from glutamate neurotoxicity and that AA may be one potential treatment for RGC loss in a variety of eye diseases, including glaucoma.

cGMP-induced presynaptic depression and postsynaptic facilitation at glutamatergic synapses in visual cortex.

The mechanisms by which the intracellular messenger cGMP can modulate synaptic efficacy remain poorly understood. Here we report that cGMP, acting through cGMP-dependent protein kinase (PKG), has multiple rapid and reversible effects on synaptic transmission in slices and cultures of rodent visual cortex. Extracellular application of the membrane permeable cGMP analog 8-bromoguanosine-3',5'-cyclic monophosphate (8-Br-cGMP) and the PKG specific activator beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate sp-isomer (Sp-8-Br-PET-cGMPS) reduced stimulus-evoked EPSPs in slices. In cortical cultures, both analogs reduced the frequency of spontaneous EPSCs, but not their amplitude. In both slices and cultures, intracellular perfusion of the postsynaptic neurons with a pseudosubstrate inhibitory peptide specific for PKG had no effect on the reduction in EPSPs and EPSCs, indicating that the inhibition occurred at presynaptic sites. Whole-cell calcium currents in cultured cortical neurons were also reduced by both analogs, which may account for the effect on synaptic release. To determine whether cGMP was also acting at postsynaptic sites, we applied exogenous kainate/AMPA and NMDA to the recorded cells directly. cGMP and its analogs showed little effect on the postsynaptic kainate/AMPA responses but produced a dramatic enhancement of NMDA responses. cGMP-induced NMDA potentiation was prevented by the specific PKG inhibitory peptide infused into the postsynaptic cell. In summary, cGMP, acting through PKG, had depressive presynaptic and facilitatory postsynaptic actions at excitatory synapses in the visual cortex. We suggest that these opposing actions may be useful for altering the balance of synaptic inputs to cortical neurons in ways that enhance signals important for synaptic facilitation and neuronal plasticity.