Dry eye syndrome (DES) is an age-related condition increasingly detected in younger people of risk groups, including patients who underwent ocular surgery or long-term general anesthesia. Being a multifactorial disease, it is characterized by oxidative stress in the cornea and commonly complicated by ocular surface inflammation. Polyetiologic DES is responsive to SkQ1, a mitochondria-targeted antioxidant suppressing age-related changes in the ocular tissues. Here, we demonstrate safety and efficacy of topical administration of SkQ1 at a dosage of 7.5 μM for the prevention of general anesthesia-induced DES in rabbits. The protective action of SkQ1 improves clinical state of the ocular surface by inhibiting apoptotic and prenecrotic changes in the corneal epithelium. The underlying mechanism involves the suppression of the oxidative stress supported by the stimulation of intrinsic antioxidant activity and the activity of antioxidant enzymes, foremost glutathione peroxidase and glutathione reductase, in the cornea. Furthermore, SkQ1 increases antioxidant activity and stability of the tear film and produces anti-inflammatory effect exhibited as downregulation of TNF-α and IL-6 and pronounced upregulation of IL-10 in tears. Our data suggest novel features of SkQ1 and point to its feasibility in patients with DES and individuals at risk for the disease including those subjected to general anesthesia.

Neuronal calcium sensor-1 (NCS-1) protein is abundantly expressed in the central nervous system and retinal neurons, where it regulates many vital processes such as synaptic transmission. It coordinates three calcium ions by EF-hands 2-4, thereby transducing Ca2+ signals to a wide range of protein targets, including G protein-coupled receptors and their kinases. Here, we demonstrate that NCS-1 also has Zn2+-binding sites, which affect its structural and functional properties upon filling. Fluorescence and circular dichroism experiments reveal the impact of Zn2+ binding on NCS-1 secondary and tertiary structure. According to atomic absorption spectroscopy and isothermal titration calorimetry studies, apo-NCS-1 has two high-affinity (4 × 106 M-1) and one low-affinity (2 × 105 M-1) Zn2+-binding sites, whereas Mg2+-loaded and Ca2+-loaded forms (which dominate under physiological conditions) bind two zinc ions with submicromolar affinity. Metal competition analysis and circular dichroism studies suggest that Zn2+-binding sites of apo- and Mg2+-loaded NCS-1 overlap with functional EF-hands of the protein. Consistently, high Zn2+ concentrations displace Mg2+ from the EF-hands and decrease the stoichiometry of Ca2+ binding. Meanwhile, one of the EF-hands of Zn2+-saturated NCS-1 exhibits a 14-fold higher calcium affinity, which increases the overall calcium sensitivity of the protein. Based on QM/MM molecular dynamics simulations, Zn2+ binding to Ca2+-loaded NCS-1 could occur at EF-hands 2 and 4. The high-affinity zinc binding increases the thermal stability of Ca2+-free NCS-1 and favours the interaction of its Ca2+-loaded form with target proteins, such as dopamine receptor D2R and GRK1. In contrast, low-affinity zinc binding promotes NCS-1 aggregation accompanied by the formation of twisted rope-like structures. Altogether, our findings suggest a complex interplay between magnesium, calcium and zinc binding to NCS-1, leading to the appearance of multiple conformations of the protein, in turn modulating its functional status.

Over 100 million individuals are affected by irreversible visual impairments and blindness worldwide, while ocular diseases remain a challenging problem despite significant advances in modern ophthalmology. Development of novel drugs and drug delivery mechanisms, as well as advanced ophthalmological techniques requires experimental models including animals, capable of developing ocular diseases with similar etiology and pathology, suitable for future trials of new therapeutic approaches. Although experimental ophthalmology and visual research are traditionally performed on rodent models, these animals are often unsuitable for pre-clinical drug efficacy and safety studies, as well as for testing novel drug delivery approaches, e.g. controlled release of pharmaceuticals using intra-ocular implants. Therefore, rabbit models of ocular diseases are particularly useful in this context, since rabbits can be easily handled, while sharing more common anatomical and biochemical features with humans compared to rodents, including longer life span and larger eye size. This review provides a brief description of clinical, morphological and mechanistic aspects of the most common ocular diseases (dry eye syndrome, glaucoma, age-related macular degeneration, light-induced retinopathies, cataract and uveitis) and summarizes the diversity of current strategies for their experimental modeling in rabbits. Several applications of some of these models in ocular pharmacology and eye care strategies are also discussed.

Light-induced oxidation of lipids and proteins provokes retinal injuries and results in progression of degenerative retinal diseases, such as, for instance, iatrogenic photic maculopathies. Having accumulated over years retinal injuries contribute to development of age-related macular degeneration (AMD). Antioxidant treatment is regarded as a promising approach to protecting the retina from light damage and AMD. Here, we examine oxidative processes induced in rabbit retina by excessive light illumination with or without premedication using mitochondria-targeted antioxidant SkQ1 (10-(6’-plastoquinonyl)decyltriphenyl-phosphonium). The retinal extracts obtained from animals euthanized within 1–7 days post exposure were analyzed for H2O2, malondialdehyde (MDA), total antioxidant activity (AOA), and activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) using colorimetric and luminescence assays. Oxidation of visual arrestin was monitored by immunoblotting. The light exposure induced lipid peroxidation and H2O2 accumulation in the retinal cells. Unexpectedly, it prominently upregulated AOA in retinal extracts although SOD and GPx activities were compromised. These alterations were accompanied by accumulation of disulfide dimers of arrestin revealing oxidative stress in the photoreceptors. Premedication of the eyes with SkQ1 accelerated normalization of H2O2 levels and redox-status of lipids and proteins, contemporarily enhancing AOA and, likely, sustaining normal activity of GPx. Thus, SkQ1 protects the retina from light-induced oxidative stress and could be employed to suppress oxidative damage of proteins and lipids contributing to AMD.

NCS1 (neuronal calcium sensor-1) is a Ca(2+)-myristoyl switch protein of the NCS protein family involved in synaptic plasticity and neurotransmission via Ca(2+)-dependent regulation of dopamine D2 receptor and associated Gprotein coupled receptor kinase (GRK)-2. Overexpression of NCS1 in synaptic terminals results in accumulation of membrane-bound protein and its redundant regulatory activity associated with neurological disorders. Here, we have demonstrated that bovine photoreceptors contain NCS1 that is capable of a partially irreversible interaction with isolated photoreceptor membranes and implicated in Ca(2+)-dependent binding and regulation of GRK1 in vitro. Using NCS1- recoverin C-terminal chimeric construct (NR), it was found that the Ca(2+)-myristoyl switch of NCS1 is affected by its C-terminal segment downstream the fourth EF-loop of the protein, which is variable within the NCS family. NR retains structural stability and sensitivity to Ca(2+), but interacts with photoreceptor membranes with lower affinity in a Ca(2+)- dependent fully reversible manner and displays altered GRK1 modulation. These data combined with fluorescent probing of surface hydrophobicity of NCS1, NR and recoverin suggest that the C-terminal segment of NCS1 regulates reuptake of myristoyl group under Ca(2+)-free conditions and participates in organization of the target-binding pocket of the protein. We point out a putative role of NCS1 in photoreceptors as a modulator of GRK activity and propose targeting of the C-terminal segment of NCS1 as an appropriate way for selective suppression of excessive membrane accumulation and aberrant activity of the protein in neurons associated with central nervous system dysfunctions.