This study aimed to investigate the interactions between fingolimod, a sphingosine 1-phosphate receptor (S1PR) agonist, and melanocortin receptors 1 and 5 (MCR1, MCR5). In particular, we investigated the effects of fingolimod, a drug approved to treat relapsing-remitting multiple sclerosis, on retinal angiogenesis in a mouse model of diabetic retinopathy (DR). We showed, by a molecular modeling approach, that fingolimod can bind with good-predicted affinity to MC1R and MC5R. Thereafter, we investigated the fingolimod actions on retinal MC1Rs/MC5Rs in C57BL/6J mice. Diabetes was induced in C57BL/6J mice through streptozotocin injection. Diabetic and control C57BL/6J mice received fingolimod, by oral route, for 12 weeks and a monthly intravitreally injection of MC1R antagonist (AGRP), MC5R antagonist (PG20N), and the selective S1PR1 antagonist (Ex 26). Diabetic animals treated with fingolimod showed a decrease of retinal vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2), compared to diabetic control group. Fingolimod co-treatment with MC1R and MC5R selective antagonists significantly (p < 0.05) increased retinal VEGFR1, VEGFR2, and VEGFA levels compared to mice treated with fingolimod alone. Diabetic animals treated with fingolimod plus Ex 26 (S1PR1 selective blocker) had VEGFR1, VEGFR2, and VEGFA levels between diabetic mice group and the group of diabetic mice treated with fingolimod alone. This vascular protective effect of fingolimod, through activation of MC1R and MC5R, was evidenced also by fluorescein angiography in mice. Finally, molecular dynamic simulations showed a strong similarity between fingolimod and the MC1R agonist BMS-470539. In conclusion, the anti-angiogenic activity exerted by fingolimod in DR seems to be mediated not only through S1P1R, but also by melanocortin receptors.

Split intein-mediated protein trans-splicing expands AAV transfer capacity, thus overcoming the limited AAV cargo. However, non-mammalian inteins persist as trans-splicing by-products, and this could raise safety concerns for AAV intein clinical applications. In this study, we tested the ability of several degrons to selectively decrease levels of inteins after protein trans-splicing and found that a version of E. coli dihydrofolate reductase, which we have shortened to better fit into the AAV vector, is the most effective. We show that subretinal administration of AAV intein armed with this short degron is both safe and effective in a mouse model of Stargardt disease (STGD1), which is the most common form of inherited macular degeneration in humans. This supports the use of optimized AAV intein for gene therapy of both STGD1 and other conditions that require transfer of large genes.

Background: Diabetic retinopathy (DR) is a neurovascular disease, characterized by a deficiency of brain-derived neurotrophic factor (BDNF), a regulator of autophagy. Beta-hydroxybutyrate (BHB), previously reported as a protective agent in DR, has been associated with BDNF promotion. Here, we investigated whether systemic BHB affects the retinal levels of BDNF and local autophagy in diabetic mice with retinopathy; Methods: C57BL/6J mice were administered with intraperitoneal (i.p.) streptozotocin (STZ) (75 mg/kg) injection to develop diabetes. After 2 weeks, they received i.p. injections of BHB (25–50–100 mg/kg) twice a week for 10 weeks. Retinal samples were collected in order to perform immunofluorescence, Western blotting, and ELISA analysis; Results: BHB 50 mg/kg and 100 mg/kg significantly improved retinal BDNF levels (p < 0.01) in diabetic mice. This improvement was negatively associated with autophagosome–lysosome formations (marked by LC3B and ATG14) and to higher levels of connexin 43 (p < 0.01), a marker of cell integrity. Moreover, BHB administration significantly reduced M1 microglial activation and autophagy (p < 0.01); Conclusions: The systemic administration of BHB in mice with DR improves the retinal levels of BDNF, with the consequent reduction of the abnormal microglial autophagy. This leads to retinal cell safety through connexin 43 restoration.

The most frequent retinal diseases, such as diabetic retinopathy, age-related macular degeneration and posterior uveitis, are underlined by oxidative stress or aging-induced retinal inflammation, which contributes to vision impairing or loss. Resolution of inflammation is emerging as a critical phase able to counteract the inflammatory process leading to the progression of retinal damage. Particularly, pro-resolving mediators (PMs) play a key role in the modulation of inflammatory exudates and could be considered a new target to be investigated in different inflammatory-autoimmune pathologies. Here, we highlight the most recent studies concerning the role of the main PMs (lipoxins, resolvins, prtectins, maresins and annexins) in retinal inflammation, in order to collect the best evidence in the field of inflammatory retinal damage resolution and to propose novel pharmacological approaches in the management of the most common retinal diseases.

(1) Background: The pro-resolving lipid mediator Resolvin D1 (RvD1) has already shown protective effects in animal models of diabetic retinopathy. This study aimed to investigate the retinal levels of RvD1 in aged (24 months) and younger (3 months) Balb/c mice, along with the activation of macro- and microglia, apoptosis, and neuroinflammation. (2) Methods: Retinas from male and female mice were used for immunohistochemistry, immunofluorescence, transmission electron microscopy, Western blotting, and enzyme-linked immunosorbent assays. (3) Results: Endogenous retinal levels of RvD1 were reduced in aged mice. While RvD1 levels were similar in younger males and females, they were markedly decreased in aged males but less reduced in aged females. Both aged males and females showed a significant increase in retinal microglia activation compared to younger mice, with a more marked reactivity in aged males than in aged females. The same trend was shown by astrocyte activation, neuroinflammation, apoptosis, and nitrosative stress, in line with the microglia and Müller cell hypertrophy evidenced in aged retinas by electron microscopy. (4) Conclusions: Aged mice had sex-related differences in neuroinflammation and apoptosis and low retinal levels of endogenous RvD1.

The genome-wide activity of transcription factors (TFs) on multiple regulatory elements precludes their use as gene-specific regulators. Here we show that ectopic expression of a TF in a cell-specific context can be used to silence the expression of a specific gene as a therapeutic approach to regulate gene expression in human disease. We selected the TF Krüppel-like factor 15 ( KLF15 ) based on its putative ability to recognize a specific DNA sequence motif present in the rhodopsin ( RHO ) promoter and its lack of expression in terminally differentiated rod photoreceptors (the RHO -expressing cells). Adeno-associated virus (AAV) vector–mediated ectopic expression of KLF15 in rod photoreceptors of pigs enables Rho silencing with limited genome-wide transcriptional perturbations. Suppression of a RHO mutant allele by KLF15 corrects the phenotype of a mouse model of retinitis pigmentosa with no observed toxicity. Cell-specific-context conditioning of TF activity may prove a novel mode for somatic gene–targeted manipulation.