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Cystic fibrosis (CF) is a hereditary disease mostly related to ΔF508 CFTR mutation causing a proteinopathy that is characterized by multiple organ dysfunction, primarily lungs chronic inflammation, and infection. Defective autophagy and accumulation of the inflammatory lipid ceramide have been proposed as therapeutic targets. Accumulation of lipids and cholesterol was reported in the airways of CF patients, together with altered triglycerides and cholesterol levels in plasma, thus suggesting a disease-related dyslipidemia. Myriocin, an inhibitor of sphingolipids synthesis, significantly reduces inflammation and activates TFEB-induced response to stress, enhancing fatty acids oxidation and promoting autophagy. Myriocin ameliorates the response against microbial infection in CF models and patients' monocytes. Here we show that CF broncho-epithelial cells exhibit an altered distribution of intracellular lipids. We demonstrated that lipid accumulation is supported by an enhanced synthesis of fatty acids containing molecules and that Myriocin is able to reduce such accumulation. Moreover, Myriocin modulated the transcriptional profile of CF cells in order to restore autophagy, activate an anti-oxidative response, stimulate lipid metabolism and reduce lipid peroxidation. Moreover, lipid storage may be altered in CF cells, since we observed a reduced expression of lipid droplets related proteins named perilipin 3 and 5 and seipin. To note, Myriocin up-regulates the expression of genes that are involved in lipid droplets biosynthesis and maturation. We suggest that targeting sphingolipids de novo synthesis may counteract lipids accumulation by modulating CF altered transcriptional profile, thus restoring autophagy and lipid metabolism homeostasis., (Copyright © 2021. Published by Elsevier Inc.)

Background/aims: Cystic Fibrosis (CF) is an inherited disease associated with a variety of mutations affecting the CFTR gene. A deletion of phenylalanine 508 (F508) affects more than 70% of patients and results in unfolded proteins accumulation, originating a proteinopathy responsible for inflammation, impaired trafficking, altered metabolism, cholesterol and lipids accumulation, impaired autophagy at the cellular level. Lung inflammation has been extensively related to the accumulation of the lipotoxin ceramide. We recently proved that inhibition of ceramide synthesis by Myriocin reduces inflammation and ameliorates the defence response against pathogens infection, which is downregulated in CF. Here, we aim at demonstrating the mechanisms of Myriocin therapeutic effects in Cystic Fibrosis broncho-epithelial cells., Methods: The effect of Myriocin treatment, on F508-CFTR bronchial epithelial cell line IB3-1 cells, was studied by evaluating the expression of key proteins and genes involved in autophagy and lipid metabolism, by western blotting and real time PCR. Moreover, the amount of glycerol-phospholipids, triglycerides, and cholesterols, sphingomyelins and ceramides were measured in treated and untreated cells by LC-MS. Finally, Sptlc1 was transiently silenced and the effect on ceramide content, autophagy and transcriptional activities was evaluated as above mentioned., Results: We demonstrate that Myriocin tightly regulates metabolic function and cell resilience to stress. Myriocin moves a transcriptional program that activates TFEB, major lipid metabolism and autophagy regulator, and FOXOs, central lipid metabolism and anti-inflammatory/anti-oxidant regulators. The activity of these transcriptional factors is associated with the induction of PPARs nuclear receptors activity, whose targets are genes involved in lipid transport compartmentalization and oxidation. Transient silencing of SPTCL1 recapitulates the effects induced by Myriocin., Conclusion: Cystic Fibrosis bronchial epithelia accumulate lipids, exacerbating inflammation. Myriocin administration: i) activates the transcriptions of genes involved in enhancing autophagy-mediated stress clearance; ii) reduces the content of several lipid species and, at the same time, iii) enhances mitochondrial lipid oxidation. Silencing the expression of Sptlc1 reproduces Myriocin induced autophagy and transcriptional activities, demonstrating that the inhibition of sphingolipid synthesis drives a transcriptional program aimed at addressing cell metabolism towards lipid oxidation and at exploiting autophagy mediated clearance of stress. We speculate that regulating sphingolipid de novo synthesis can relieve from chronic inflammation, improving energy supply and anti-oxidant responses, indicating an innovative therapeutic strategy for CF., Competing Interests: All the authors of this manuscript have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Myriocin is an antibiotic derived from Mycelia sterilia, and is a potent inhibitor of serine palmitoyltransferase, the enzyme involved in the first step of sphingosine synthesis. Myriocin, inhibiting ceramide synthesis, has a great potential for treatment of diseases characterized by high ceramide levels in affected tissues, such as retinitis pigmentosa (RP). Drug delivery to the retina is a challenging task, which is generally by-passed through intravitreal injection, that represents a risky invasive procedure. We, therefore, developed and characterized an ophthalmic topical nanotechnological formulation based on a nanostructured lipid carrier (NLC) and containing myriocin. The ocular distribution of myriocin in the back of the eye was assessed both in rabbits and mice using LC-MS/MS. Moreover, rabbit retinal sphingolipid and ceramides levels, after myriocin-NLC (Myr-NLC) eye drops treatment, were assessed. The results demonstrated that Myr-NLC formulation is well tolerated and provided effective levels of myriocin in the back of the eye both in rabbits and mice. We found that Myr-NLC eye drops treatment was able to significantly decrease retinal sphingolipid levels. In conclusion, these data suggest that the Myr-NLC ophthalmic formulation is suitable for pharmaceutical development and warrants further clinical evaluation of this eye drops for the treatment of RP.