Sphingomyelin synthases in depression and autophagy

Sphingolipids are a complex family of bioactive lipids with ceramide as a central component. Sphingomyelin synthases (SMS) play a critical role in sphingolipid metabolism, catalysing the conversion of ceramide and phosphatidylcholine into sphingomyelin and diacylglycerol. Mis-balances in the ceramide/sphingomyelin system have been linked to Major depressive disor-der (MDD), a debilitating and severe psychiatric disease with a combination of various affec-tive, social, and somatic symptoms. The pathological mechanisms remain largely unknown. Further, current antidepressants have notable limitations, including delayed action and a high rate of relapse, highlighting a pressing need for the development of efficient and fast-acting antidepressants. Numerous antidepressants functionally inhibit acid sphingomyelinase (ASM), which catalyses the reverse reaction to SMS, and the presence of ASM is required for the an-tidepressant action of these drugs. Recent studies in mice have revealed that pharmacological inhibition of SMS rapidly improves depression-like behaviour by inducing neurogenesis and activating autophagy. However, whether these effects are due to the inhibition of one or both SMS isoforms (SMS1 and SMS2) remains unknown. My PhD thesis thus aimed to establish a reliable in vitro assay to assess SMS activity, to elucidate the involvement of SMS1 and SMS2 in depression and to determine their role in the autophagy-dependent antidepressant effect. After developing an in vitro assay for SMS activity in cell and tissue lysates as well as in human blood cell samples based on a fluorescently labelled substrate, the enzyme was characterized biochemically. SMS activity was very sensitive to the presence of detergents and independent of divalent cations, and it was rather inhibited by most tested metal ions. The specificity of the assay for SMS was validated by successfully detecting overexpression or siRNA-mediated downregulation of SMS1 or SMS2. To investigate whether SMS is involved in depression in humans, the activity and expression of SMS in leukocytes from patients experiencing a major depressive episode were compared to healthy controls. We found a sex-independent higher expression of all SMS genes (SGMS1, SGMS1, SAMD8) under the pathological conditions. In addition, higher SMS enzymatic activity in blood cells was observed in female, but not male, depressed patients compared to controls. Moreover, SMS activity strongly positively correlated with the activity of lysosomal ASM, sug-gesting a coregulation of both enzymes to maintain ceramide levels. Finally, to study the specific roles of SMS isoforms in autophagy, we used siRNAs to specifically downregulate SMS isoforms in human neuroglioma H4 cells as a model. The in vitro studies showed that downregulation of SMS1, but not SMS2 or both, led to an accumulation of the autophagic marker LC3-II in the presence of Bafilomycin A1 compared to control cells, indicat-ing an increased autophagic flux. In addition, SMS1 downregulation resulted in an increased expression of SQSTM1 (p62) as well as ULK1 and PIK3C3, coding for the kinases involved in the early initiation steps of the autophagy process. Protein levels of ULK1 were increased after SMS1 knock-down, but the phosphorylation ratio at its active sites was reduced. Beclin 1 phos-phorylation was increased after SMS1 but not SMS2 downregulation, suggesting an active sta-tus of the PIK3C3 complex. Moreover, we demonstrated that phosphorylation of Beclin 1 was mediated by ULK1, since it was impaired after pharmacological inhibition of ULK1 by SBI- 0206965 or ULK-101. Sphingolipidomic analysis after SMS1 or SMS2 downregulation resulted in the expected decreased total sphingomyelin/ceramide ratio, but the levels of most individ-ual sphingomyelin or ceramide species were not significantly altered. Interestingly, SMS downregulation also elevated levels of glucosylceramide species, raising the possibility of the involvement of other sphingolipids different from ceramide in the autophagy triggering medi-ated by SMS1 knock-down. Altogether, we developed an assay to measure SMS enzymatic activity in a broad variety of samples, our clinical results support the hypothesis of increased SMS activity in depression and the in vitro data indicate a specific involvement of SMS1 in autophagy induction. Thereby, this study not only provides further insights into the relationship between sphingolipids and depression, but also identifies SMS1 as a potential pharmacological target for the design of fast-acting antidepressants.