Identificazione di nuovi meccanismi coinvolti nella disfunzione dell’Epitelio Pigmentato Retinico come bersagli terapeutici per la Degenerazione Maculare Legata all’Età.

This project take place in a research field that aims to deepen the knowledge of human retinal degenerations. In this wide field, the present study focuses on the study of AMD, which is considered a multifactorial disorder caused by both environmental and genetic risk factors. AMD incidence increases with age, and it is going to raise dramatically due to life expectancy increase in the modern age. In fact, according to the World Health Organization (WHO), over two billion people worldwide suffer from vision-related problems. AMD could be classified into two main forms with different features: dry AMD and wet (or exudative) AMD. The available therapies to date are not resolutive; thus, the research on this topic is continuously evolving to find new targets for the development of effective treatments. During AMD, retinal antioxidant machinery is impaired, leading to oxidative stress burden, which is therefore considered one of the main risk factors for this pathology. In this project, we focused our attention on the degenerative events occurring in the Retinal Pigment Epithelium (RPE) during AMD and the analysis of possible protective molecules. RPE plays a key role in the maintenance of retinal homeostasis through its several functions; however, during AMD, oxidative stress triggers several RPE alterations which culminate in a failure of RPE performance and its consequent degeneration. On these bases, here we investigated the potential protective outcomes of Curcumin and L-DOPA on RPE cells challenged with oxidative stress. Both these molecules have already shown protective effects against retinal degenerations; thus, we aimed at identifying new mechanisms of action of these molecules. We analysed two main aspects linked to RPE dysfunction during AMD: cellular homeostasis and melanogenesis. In particular, we provided new understanding on the dose-dependent role of Curcumin; We found that low concentrations maintain RPE cells homeostasis while high concentrations caused a harmful stimulus to the cells, inducing proliferation arrest and autophagy activation. These data support the application of Curcumin for ocular pathologies characterized by different features. Furthermore, we identified a new mechanism of action of curcumin, demonstrating that the low concentrations prevented oxidative stress-induced cell death by inhibiting the activity of SIRT1, an enzyme involved in AMD progression. In the second part of the project, we evaluated the role of L-DOPA on cellular homeostasis and melanogenesis. L-DOPA was able to prevent oxidative stress-induced cell death, acting on different pathways including mitochondrial homeostasis and autophagy. We demonstrated that the protective effect of L-DOPA is related to the activation of its specific receptor on RPE cells, GPR143, as also demonstrated by Intracellular Calcium increase. Furthermore, the activation of GPR143 by L-DOPA induced a downstream event related to the the modulation of Vascular Endothelial Growth Factor (VEGF) and Pigmented Epithelium-derived factor (PEDF), the main growth factors involved in RPE function. L-DOPA administration was also associated with the up-regulation of Tyrosinase, demonstrating the role of L-DOPA in enhancing melanogenesis process. In the last part of this project, through an in silico study, we also provided evidence of the interaction of L-DOPA with another receptor involved in melanogenesis, Melanocortin 1 receptor (MC1R). All together, these results provided new understanding of RPE degenerative events caused by oxidative stress and underlined new mechanisms of action of Curcumin and L-DOPA, two promising molecules for the treatment of AMD.