ИСТИНА

В проекте планируется впервые всесторонне охарактеризовать биохимические процессы, лежащие в основе цинкзависимой нейродегенерации сетчатки, на уровне пациентов, с использованием животных и клеточных моделей, а также в экспериментах in vitro и in silico.

The retina is the neuronal tissue of the eye responsible for the reception and transduction of the visual signal. Damage to retinal neurons due to various reasons underlies the development of glaucoma and age-related macular degeneration (AMD), common ophthalmic diseases leading to irreversible loss of vision. Neurodegenerative processes in these diseases are characterized by a complex etiology associated with the anatomical features and protein composition of the retina, its unique metabolic activity, photosensitivity, immune privilege, and other specific features. One of these features of the retina is the high content of zinc ions. Zinc is one of the main physiological metals, the correct homeostasis of which is critical for the normal functionality of retinal neurons. The lack of zinc, which possesses antioxidant and anti-inflammatory activity, leads to damage to retinal pigment epithelium and photoreceptor neurons in AMD. At the same time, an increase in the concentration of so-called mobile or chelatable zinc in the retina has recently been identified as a key factor causing the degeneration of ganglion neurons in glaucoma, along with oxidative stress, excitotoxicity, and other processes. It is believed that being bound to proteins with different affinities, zinc can perform not only a structural role and the role of a cofactor in enzymes but also a signaling function in the cell. Moreover, at high concentrations, zinc can cause protein aggregation thereby exhibiting cytotoxic effects. Protein targets and molecular mechanisms mediating the neuroprotective and neurotoxic effects of zinc in the retina remain largely unknown. This project is aimed at the comprehensive characterization of the biochemical processes underlying zinc-dependent neurodegeneration of the retina, in human studies, using animal and cellular models, as well as in vitro and in silico experiments. Using samples obtained from patients with various stages of primary open-angle glaucoma, we will conduct a biochemical analysis of aqueous humor (AH). This intraocular fluid provides nutrition and immunity to the eye tissues and its abnormal circulation contributes to the development of glaucoma. AH is in direct contact with the retina (the retina is separated from the bloodstream by the blood-retinal barrier), and the biochemical composition of the AH can reflect the state of this tissue. In this project, we will identify changes in the AH content of zinc and its low molecular weight chelators, associated with the development of glaucoma. In addition, we will characterize other metabolomic and lipidomic changes in AH, including alterations in the content of polyunsaturated fatty acids and their derivatives oxlipins, lipid mediators of oxidative stress, and inflammation. Using the experimental (animal) models of glaucoma and AMD developed in our previous studies, we will determine corresponding changes in the molecular composition of AH. Furthermore, it is planned to characterize the zinc-dependent proteome of the retina and determine changes in the content of zinc-binding retinal proteins in these diseases. We also plan to identify correlations between the detected biochemical effects, the development of pathomorphological changes in the retina, and alterations in its electrophysiological activity in glaucoma and AMD. We will also predict the potential zinc-binding proteins of the retina using novel methods of structural bioinformatics by analyzing the resolved structures of all retinal proteins present in PDB. Using bioinformatic analysis, we will systematize the identified and predicted zinc-dependent proteins and select functionally significant candidates for their further analysis. It is planned to carry out cloning, expression, and purification of the selected proteins and characterization of their zinc-binding and other structural and functional properties in vitro. Finally, using a cellular model of the retina, retinoblastoma Y79 cell line, we will characterize the protective and cytotoxic effects of zinc, and investigate the role of the identified zinc-binding proteins as mediators of these effects. Based on the data obtained, methods of neuroprotective therapy of the retina will be proposed and tested in animal models of glaucoma and AMD using histological, electrophysiological, biochemical, and other methods and approaches. Overall, the proposed studies will establish for the first time biochemical molecules mediating the protective and cytotoxic effects of zinc in the retina, the targeting of which can be considered as a promising therapy for glaucoma and AMD.