We recently demonstrated that 48 h exposure of primary human bronchial epithelial (hBE) cells, obtained from both CF (F508del homozygous) and non-CF subjects, to the triple drug combination Elexacaftor/Tezacaftor/Ivacaftor (ETI) results in a CFTR genotype-independent modulation of the de novo synthethic pathway of sphingolipids, with an accumulation of dihydroceramides (dHCer). Since dHCer are converted into ceramides (Cer) by the action of a delta-4 sphingolipid desaturase (DEGS) enzyme, we aimed to better understand this off-target effect of ETI (i.e., not related to CFTR rescue) METHODS: hBE cells, both F508del and wild-type, were cultured to create fully differentiated bronchial epithelia. We analyzed Cer and dHCer using an LC-MS based method previously developed by our lab. DEGS expression levels in differentiated hBE cells lysates were quantified by western blot analysis. We demonstrated that 1) dHCer accumulate in hBE with time following prolonged ETI exposure, that 2) similar inhibition occurs in wild-type primary human hepatocytes and that 3) this does not result in an alteration of DEGS expression. We then proved that 4) ETI is a direct inhibitor of DEGS, that 5) Tezacaftor is the molecule responsible for this effect, that 6) the inhibition is concentration dependent. Finally, after repeated oral administration of ETI to naïve, non-CF, mice, we observed a slight accumulation of dHCer in the brain. We believe that further investigations on Tezacaftor should be envisaged, particularly for the use of ETI during pregnancy, breastfeeding and in the early stages of development. DEGS dysfunction and dHCer accumulation causes impairment in the development of the nervous system, due to a derangement in myelin formation and maintenance.

Cystic fibrosis (CF) is the most common inherited, life‐limiting disorder in Caucasian populations. It is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which lead to an impairment of protein expression and/or function. CFTR is a chloride/bicarbonate channel expressed at the apical surface of epithelial cells of different organs. Nowadays, more than 2100 CFTR genetic variants have been described, but not all of them cause CF. However, around 80–85% of the patients worldwide are characterized by the presence, at least in one allele, of the mutation F508del. CFTR mutations cause aberrant hydration and secretion of mucus in hollow organs. In the lungs, this condition favors bacterial colonization, allowing the development of chronic infections that lead to the onset of the CF lung disease, which is the main cause of death in patients. In recent years, evidence has reported that CFTR loss of function is responsible for alterations in a particular class of bioactive lipids, called sphingolipids (SL). SL are ubiquitously present in eukaryotic cells and are mainly asymmetrically located within the external leaflet of the plasma membrane, where they organize specific platforms capable of segregating a selected number of proteins. CFTR is associated with these platforms that are fundamental for its functioning. Considering the importance of SL in CFTR homeostasis, we attempt here to provide a critical overview of the literature to determine the role of these lipids in channel stability and activity, and whether their modulation in CF could be a target for new therapeutic approaches.

Background: GM3 Synthase Deficiency (GM3SD) is a rare autosomal recessive neurodevelopmental disease characterized by recurrent seizures and neurological deficits. The disorder stems from mutations in the ST3GAL5 gene, encoding GM3 synthase (GM3S), a key enzyme in ganglioside biosynthesis. While enzyme deficiencies affecting ganglioside catabolism are well-documented, the consequences of impaired ganglioside biosynthesis remain less explored. Methods: To investigate GM3SD, we used a Human Embryonic Kidney 293-T (HEK293-T) knockout (KO) cell model generated via CRISPR/Cas9 technology. Lipid composition was assessed via high-performance thin-layer chromatography (HPTLC); glycohydrolase activity in lysosomal and plasma membrane (PM) fractions was enzymatically analyzed. Lysosomal homeostasis was evaluated through protein content analysis and immunofluorescence, and cellular bioenergetics was measured using a luminescence-based assay. Results: Lipidome profiling revealed a significant accumulation of lactosylceramide (LacCer), the substrate of GM3S, along with increased levels of monosialyl-globoside Gb5 (MSGb5), indicating a metabolic shift in glycosphingolipid biosynthesis. Lipid raft analysis revealed elevated cholesterol levels, which may impair microdomain fluidity and signal transduction. Furthermore, altered activity of lysosomal and plasma membrane (PM)-associated glycohydrolases suggests secondary deregulation of glycosphingolipid metabolism, potentially contributing to abnormal lipid patterns. In addition, we observed increased lysosomal mass, indicating potential lysosomal homeostasis dysregulation. Finally, decreased adenosine triphosphate (ATP) levels point to impaired cellular bioenergetics, emphasizing the metabolic consequences of GM3SD. Conclusions: Together, these findings provide novel insights into the molecular alterations associated with GM3SD and establish the HEK293-T KO model as a promising platform for evaluating potential therapeutic strategies.