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Insulin-mechanistic target of rapamycin (mTOR) signaling drives anabolic growth during organismal development; its late-life dysregulation contributes to aging and limits lifespans. Age-related regulatory mechanisms and functional consequences of insulin-mTOR remain incompletely understood. Here, we identify LPD-3 as a megaprotein that orchestrates the tempo of insulin-mTOR signaling during C. elegans aging. We find that an agonist insulin, INS-7, is drastically overproduced from early life and shortens lifespan in lpd-3 mutants. LPD-3 forms a bridge-like tunnel megaprotein to facilitate non-vesicular cellular lipid trafficking. Lipidomic profiling reveals increased hexaceramide species in lpd-3 mutants, accompanied by up-regulation of hexaceramide biosynthetic enzymes, including HYL-1. Reducing the abundance of HYL-1, insulin receptor/DAF-2 or mTOR/LET-363, normalizes INS-7 levels and rescues the lifespan of lpd-3 mutants. LPD-3 antagonizes SINH-1, a key mTORC2 component, and decreases expression with age. We propose that LPD-3 acts as a megaprotein brake for organismal aging and that its age-dependent decline restricts lifespan through the sphingolipid-hexaceramide and insulin-mTOR pathways.

Biological membranes consist of a lipid bilayer in which integral membrane proteins are embedded. Based on the compositional complexity of the lipid species found in membranes, and on their specific and selective interactions with membrane proteins, we recently suggested that membrane bilayers can be best described as "finely-tuned molecular machines." We now discuss one such set of lipid-protein interactions by describing a negative feedback mechanism operating in the de novo sphingolipid biosynthetic pathway, which occurs in the membrane of the endoplasmic reticulum, and describe the atomic interactions between the first enzyme in the pathway, namely serine palmitoyl transferase, and the product of the fourth enzyme in the pathway, ceramide. We explore how hydrogen-bonding and hydrophobic interactions formed between Asn13 and Phe63 in the serine palmitoyl transferase complex and ceramide can influence the ceramide content of the endoplasmic reticulum. This example of finely-tuned biochemical interactions raises intriguing mechanistic questions about how sphingolipids and their biosynthetic enzymes could have evolved, particularly in light of their metabolic co-dependence. [ABSTRACT FROM AUTHOR]

The synapse is a piece of information transfer machinery replacing the electrical conduction of nerve impulses at the end of the neuron. Like many biological mechanisms, its functioning is heavily affected by time constraints. The solution selected by evolution is based on chemical communication that, in theory, cannot compete with the speed of nerve conduction. Nevertheless, biochemical and biophysical compensation mechanisms mitigate this intrinsic weakness: (i) through the high concentrations of neurotransmitters inside the synaptic vesicles; (ii) through the concentration of neurotransmitter receptors in lipid rafts, which are signaling platforms; indeed, the presence of raft lipids, such as gangliosides and cholesterol, allows a fine tuning of synaptic receptors by these lipids; (iii) through the negative electrical charges of the gangliosides, which generate an attractive (for cationic neurotransmitters, such as serotonin) or repulsive (for anionic neurotransmitters, such as glutamate) electric field. This electric field controls the flow of glutamate in the tripartite synapse involving pre- and post-synaptic neurons and the astrocyte. Changes in the expression of brain gangliosides can disrupt the functioning of the glutamatergic synapse, causing fatal diseases, such as Rett syndrome. In this review, we propose an in-depth analysis of the role of gangliosides in the glutamatergic synapse, highlighting the primordial and generally overlooked role played by the electric field of synaptic gangliosides. [ABSTRACT FROM AUTHOR]

Background: Approximately 25–30% of patients with acute myeloid leukemia (AML) have FMS-like receptor tyrosine kinase-3 (FLT3) mutations that contribute to disease progression and poor prognosis. Prolonged exposure to FLT3 tyrosine kinase inhibitors (TKIs) often results in limited clinical responses due to diverse compensatory survival signals. Therefore, there is an urgent need to elucidate the mechanisms underlying FLT3 TKI resistance. Dysregulated sphingolipid metabolism frequently contributes to cancer progression and a poor therapeutic response. However, its relationship with TKI sensitivity in FLT3-mutated AML remains unknown. Thus, we aimed to assess mechanisms of FLT3 TKI resistance in AML. Methods: We performed lipidomics profiling, RNA-seq, qRT-PCR, and enzyme-linked immunosorbent assays to determine potential drivers of sorafenib resistance. FLT3 signaling was inhibited by sorafenib or quizartinib, and SPHK1 was inhibited by using an antagonist or via knockdown. Cell growth and apoptosis were assessed in FLT3-mutated and wild-type AML cell lines via Cell counting kit-8, PI staining, and Annexin-V/7AAD assays. Western blotting and immunofluorescence assays were employed to explore the underlying molecular mechanisms through rescue experiments using SPHK1 overexpression and exogenous S1P, as well as inhibitors of S1P2, β-catenin, PP2A, and GSK3β. Xenograft murine model, patient samples, and publicly available data were analyzed to corroborate our in vitro results. Results: We demonstrate that long-term sorafenib treatment upregulates SPHK1/sphingosine-1-phosphate (S1P) signaling, which in turn positively modulates β-catenin signaling to counteract TKI-mediated suppression of FLT3-mutated AML cells via the S1P2 receptor. Genetic or pharmacological inhibition of SPHK1 potently enhanced the TKI-mediated inhibition of proliferation and apoptosis induction in FLT3-mutated AML cells in vitro. SPHK1 knockdown enhanced sorafenib efficacy and improved survival of AML-xenografted mice. Mechanistically, targeting the SPHK1/S1P/S1P2 signaling synergizes with FLT3 TKIs to inhibit β-catenin activity by activating the protein phosphatase 2 A (PP2A)-glycogen synthase kinase 3β (GSK3β) pathway. Conclusions: These findings establish the sphingolipid metabolic enzyme SPHK1 as a regulator of TKI sensitivity and suggest that combining SPHK1 inhibition with TKIs could be an effective approach for treating FLT3-mutated AML. [ABSTRACT FROM AUTHOR]

Previous literature suggests that sphingolipids may impact systemic coagulation and platelet aggregation, thus modulating the risks of thrombotic events. The goal of this investigation was to evaluate the role of serum sphingolipids on intrinsic platelet function to assess whether pharmacologic manipulation of sphingolipid metabolites would impact platelet aggregability. C57BL/6J mice were injected with either normal saline, 1 mg/kg FTY720 (synthetic sphingosine-1-phosphate [S1P] receptor analog), or 5 mg/kg SLM6031434 (sphingosine kinase two inhibitor). Mice were sacrificed at 6 h and whole blood (WB) was collected for impedance aggregometry assessing platelet responsiveness to arachidonic acid or adenosine diphosphate. Ex vivo studies utilized WB or platelet-rich plasma that was pretreated with S1P, FTY720, amitriptyline, or d-sphingosine then analyzed by aggregability and flow cytometry for platelet and platelet-derived microvesicle characteristics. FTY720 and SLM6031434 pretreated induced similar arachidonic acid and adenosine diphosphate–mediated platelet aggregation as controls. Ex vivo WB and platelet-rich plasma treatment with S1P, FTY720, amitriptyline and d-sphingosine did not impact platelet aggregation. The percentages of CD41+, CD62P+ and CD41+/ceramide+, CD62P+/ceramide + platelets, and platelet-derived microvesicle were not significantly different between amitriptyline-treated and normal saline-treated cohorts. Sphingolipid modulating agents, such as FTY720, SLM6031434, S1P, amitriptyline, ceramide, and d-sphingosine do not appear to independently impact platelet aggregation in murine models. [ABSTRACT FROM AUTHOR]

The (patho)physiological function of the sphingolipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), and sphingosylphosphorylcholine (SPC) in articular joints during osteoarthritis (OA) is largely unknown. Therefore, we investigated the influence of these lipids on protein expression by fibroblast-like synoviocytes (FLSs) from OA knees. Cultured human FLSs (n = 7) were treated with 1 of 3 lipid species—C1P, S1P, or SPC—IL-1β, or with vehicle. The expression of individual proteins was determined by tandem mass tag peptide labeling followed by high-resolution electrospray ionization (ESI) mass spectrometry after liquid chromatographic separation (LC-MS/MS/MS). The mRNA levels of selected proteins were analyzed using RT-PCR. The 3sphingolipids were quantified in the SF of 18 OA patients using LC-MS/MS. A total of 4930 proteins were determined using multiplex MS, of which 136, 9, 1, and 0 were regulated both reproducibly and significantly by IL-1β, C1P, S1P, and SPC, respectively. In the presence of IL-1ß, all 3 sphingolipids exerted ancillary effects. Only low SF levels of C1P and SPC were found. In conclusion, the 3 lipid species regulated proteins that have not been described in OA. Our results indicate that charged multivesicular body protein 1b, metal cation symporter ZIP14, glutamine-fructose-6-P transaminase, metallothionein-1F and -2A, ferritin, and prosaposin are particularly interesting proteins due to their potential to affect inflammatory, anabolic, catabolic, and apoptotic mechanisms. [ABSTRACT FROM AUTHOR]

The COVID‐19 pandemic, caused by SARS‐CoV‐2, has had a significant worldwide impact, affecting millions of people. COVID‐19 is characterized by a heterogenous clinical phenotype, potentially involving hyperinflammation and prolonged tissue damage, although the exact underlying mechanisms are yet to be fully understood. Sphingolipid metabolites, which govern cell survival and proliferation, have emerged as key players in inflammatory signaling and cytokine responses. Given the complex metabolic pathway of sphingolipids, this study aimed to understand their potential role in the pathogenesis of COVID‐19. We conducted a comprehensive examination of sphingolipid modulations across groups classified based on disease severity, incorporating a time‐course in serum and urine samples. Several sphingolipids, including sphingosine, lactosylceramide, and hexosylceramide, emerged as promising indicators of COVID‐19 severity, as validated by correlation analyses conducted on both serum and urine samples. Other sphingolipids, such as sphingosine 1‐phosphate, ceramides, and deoxy‐dihydroceramides, decreased in both COVID‐19 patients and individuals with non‐COVID infectious diseases. This suggests that these sphingolipids are not specifically associated with COVID‐19 but rather with pathological conditions caused by infectious diseases. Our analysis of urine samples revealed elevated levels of various sphingolipids, with changes dependent on disease severity, potentially highlighting the acute kidney injury associated with COVID‐19. This study illuminates the intricate relationship between disturbed sphingolipid metabolism, COVID‐19 severity, and clinical factors. These findings provide valuable insights into the broader landscape of inflammatory diseases. [ABSTRACT FROM AUTHOR]

Sphingolipids are widely present in cell membranes and participate in biological processes such as cell signaling, cell proliferation, migration, invasion and apoptosis. Sphingolipids and their metabolites play important roles in the occurrence and development of tumors, including promoting tumor cell proliferation, migration and invasion. inhibiting tumor cell apoptosis and promoting immune escape. In recent years, the role of sphingolipids in a variety of tumors has been found. At present, sphingolipids and their metabolites. have become a research hotspot in tumor treatment. This article mainly described the role and application prospect of sphingolipids and their metabolites in the occurrence and development of breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer and other tumors. [ABSTRACT FROM AUTHOR]

Background: The current understanding of the mechanisms by which metal ion metabolism promotes the progression and drug resistance of osteosarcoma remains incomplete. This study aims to elucidate the key roles and mechanisms of genes involved in cuproptosis-related sphingolipid metabolism (cuproptosis-SPGs) in regulating the immune landscape, tumor metastasis, and drug resistance in osteosarcoma cells. Methods: This study employed multi-omics approaches to assess the impact of cuproptosis-SPGs on the prognosis of osteosarcoma patients. Lasso regression analysis was utilized to construct a prognostic model, while multivariate regression analysis was applied to identify key core genes and generate risk coefficients for these genes, thereby calculating a risk score for each osteosarcoma patient. Patients were then stratified into high-risk and low-risk groups based on their risk scores. The ESTIMATE and CIBERSORT algorithms were used to analyze the level of immune cell infiltration within these risk groups to construct the immune landscape. Single-cell analysis was conducted to provide a more precise depiction of the expression patterns of cuproptosis-SPGs among immune cell subtypes. Finally, experiments on osteosarcoma cells were performed to validate the role of the cuproptosis-sphingolipid signaling network in regulating cell migration and apoptosis. Results: In this study, seven cuproptosis-SPGs were identified and used to construct a prognostic model for osteosarcoma patients. In addition to predicting survival, the model also demonstrated reliability in forecasting the response to chemotherapy drugs. The results showed that a high cuproptosissphingolipid metabolism score was closely associated with reduced CD8 T cell infiltration and indicated poor prognosis in osteosarcoma patients. Cellular functional assays revealed that cuproptosis-SPGs regulated the LC3B/ERK signaling pathway, thereby triggering cell death and impairing migration capabilities in osteosarcoma cells. Conclusion: The impact of cuproptosis-related sphingolipid metabolism on the survival and migration of osteosarcoma cells, as well as on CD8 T cell infiltration, highlights the potential of targeting copper ion metabolism as a promising strategy for osteosarcoma patients. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is an inborn error of metabolism caused by inactivating mutations in SGPL1, the gene encoding sphingosine-1-phosphate lyase (SPL), an essential enzyme needed to degrade sphingolipids. SPLIS features include glomerulosclerosis, adrenal insufficiency, neurological defects, ichthyosis, and immune deficiency. Currently, there is no cure for SPLIS, and severely affected patients often die in the first years of life. We reported that adeno-associated virus (AAV) 9-mediated SGPL1 gene therapy (AAV-SPL) given to newborn Sgpl1 knockout mice that model SPLIS and die in the first few weeks of life prolonged their survival to 4.5 months and prevented or delayed the onset of SPLIS phenotypes. In this study, we tested the efficacy of a modified AAV-SPL, which we call AAV-SPL 2.0, in which the original cytomegalovirus (CMV) promoter driving the transgene is replaced with the synthetic CAG promoter used in several clinically approved gene therapy agents. AAV-SPL 2.0 infection of human embryonic kidney (HEK) cells led to 30% higher SPL expression and enzyme activity compared to AAV-SPL. Newborn Sgpl1 knockout mice receiving AAV-SPL 2.0 survived ≥ 5 months and showed normal neurodevelopment, 85% of normal weight gain over the first four months, and delayed onset of proteinuria. Over time, treated mice developed nephrosis and glomerulosclerosis, which likely resulted in their demise. Our overall findings show that AAV-SPL 2.0 performs equal to or better than AAV-SPL. However, improved kidney targeting may be necessary to achieve maximally optimized gene therapy as a potentially lifesaving SPLIS treatment.

Abstract Background Approximately 25–30% of patients with acute myeloid leukemia (AML) have FMS-like receptor tyrosine kinase-3 (FLT3) mutations that contribute to disease progression and poor prognosis. Prolonged exposure to FLT3 tyrosine kinase inhibitors (TKIs) often results in limited clinical responses due to diverse compensatory survival signals. Therefore, there is an urgent need to elucidate the mechanisms underlying FLT3 TKI resistance. Dysregulated sphingolipid metabolism frequently contributes to cancer progression and a poor therapeutic response. However, its relationship with TKI sensitivity in FLT3-mutated AML remains unknown. Thus, we aimed to assess mechanisms of FLT3 TKI resistance in AML. Methods We performed lipidomics profiling, RNA-seq, qRT-PCR, and enzyme-linked immunosorbent assays to determine potential drivers of sorafenib resistance. FLT3 signaling was inhibited by sorafenib or quizartinib, and SPHK1 was inhibited by using an antagonist or via knockdown. Cell growth and apoptosis were assessed in FLT3-mutated and wild-type AML cell lines via Cell counting kit-8, PI staining, and Annexin-V/7AAD assays. Western blotting and immunofluorescence assays were employed to explore the underlying molecular mechanisms through rescue experiments using SPHK1 overexpression and exogenous S1P, as well as inhibitors of S1P2, β-catenin, PP2A, and GSK3β. Xenograft murine model, patient samples, and publicly available data were analyzed to corroborate our in vitro results. Results We demonstrate that long-term sorafenib treatment upregulates SPHK1/sphingosine-1-phosphate (S1P) signaling, which in turn positively modulates β-catenin signaling to counteract TKI-mediated suppression of FLT3-mutated AML cells via the S1P2 receptor. Genetic or pharmacological inhibition of SPHK1 potently enhanced the TKI-mediated inhibition of proliferation and apoptosis induction in FLT3-mutated AML cells in vitro. SPHK1 knockdown enhanced sorafenib efficacy and improved survival of AML-xenografted mice. Mechanistically, targeting the SPHK1/S1P/S1P2 signaling synergizes with FLT3 TKIs to inhibit β-catenin activity by activating the protein phosphatase 2 A (PP2A)-glycogen synthase kinase 3β (GSK3β) pathway. Conclusions These findings establish the sphingolipid metabolic enzyme SPHK1 as a regulator of TKI sensitivity and suggest that combining SPHK1 inhibition with TKIs could be an effective approach for treating FLT3-mutated AML.

Psoriasis is a long-lasting skin condition characterized by redness and thick silver scales on the skins surface. It involves various skin cells, including keratinocytes, dendritic cells, T lymphocytes, and neutrophils. The treatments for psoriasis range from topical to systemic therapies, but they only alleviate the symptoms and do not provide a fundamental cure. Moreover, systemic treatments have the disadvantage of suppressing the entire bodys immune system. Therefore, a new treatment strategy with minimal impact on the immune system is required. Recent studies have shown that sphingolipid metabolites, particularly ceramide and sphingosine-1-phosphate (S1P), play a significant role in psoriasis. Specific S1P-S1P-receptor (S1PR) signaling pathways have been identified as crucial to psoriasis inflammation. Based on these findings, S1PR modulators have been investigated and have been found to improve psoriasis inflammation. This review will discuss the metabolic pathways of sphingolipids, the individual functions of these metabolites, and their potential as a new therapeutic approach to psoriasis.

Abstract Background Traumatic brain injury (TBI) causes neuroinflammation and can lead to long-term neurological dysfunction, even in cases of mild TBI (mTBI). Despite the substantial burden of this disease, the management of TBI is precluded by an incomplete understanding of its cellular mechanisms. Sphingolipids (SPL) and their metabolites have emerged as key orchestrators of biological processes related to tissue injury, neuroinflammation, and inflammation resolution. No study so far has investigated comprehensive sphingolipid profile changes immediately following TBI in animal models or human cases. In this study, sphingolipid metabolite composition was examined during the acute phases in brain tissue and plasma of mice following mTBI. Methods Wildtype mice were exposed to air-blast-mediated mTBI, with blast exposure set at 50-psi on the left cranium and 0-psi designated as Sham. Sphingolipid profile was analyzed in brain tissue and plasma during the acute phases of 1, 3, and 7 days post-TBI via liquid-chromatography-mass spectrometry. Simultaneously, gene expression of sphingolipid metabolic markers within brain tissue was analyzed using quantitative reverse transcription-polymerase chain reaction. Significance (P-values) was determined by non-parametric t-test (Mann–Whitney test) and by Tukey’s correction for multiple comparisons. Results In post-TBI brain tissue, there was a significant elevation of 1) acid sphingomyelinase (aSMase) at 1- and 3-days, 2) neutral sphingomyelinase (nSMase) at 7-days, 3) ceramide-1-phosphate levels at 1 day, and 4) monohexosylceramide (MHC) and sphingosine at 7-days. Among individual species, the study found an increase in C18:0 and a decrease in C24:1 ceramides (Cer) at 1 day; an increase in C20:0 MHC at 3 days; decrease in MHC C18:0 and increase in MHC C24:1, sphingomyelins (SM) C18:0, and C24:0 at 7 days. Moreover, many sphingolipid metabolic genes were elevated at 1 day, followed by a reduction at 3 days and an absence at 7-days post-TBI. In post-TBI plasma, there was 1) a significant reduction in Cer and MHC C22:0, and an increase in MHC C16:0 at 1 day; 2) a very significant increase in long-chain Cer C24:1 accompanied by significant decreases in Cer C24:0 and C22:0 in MHC and SM at 3 days; and 3) a significant increase of C22:0 in all classes of SPL (Cer, MHC and SM) as well as a decrease in Cer C24:1, MHC C24:1 and MHC C24:0 at 7 days. Conclusions Alterations in sphingolipid metabolite composition, particularly sphingomyelinases and short-chain ceramides, may contribute to the induction and regulation of neuroinflammatory events in the early stages of TBI, suggesting potential targets for novel diagnostic, prognostic, and therapeutic strategies in the future.

Podocyte health is vital for maintaining proper glomerular filtration in the kidney. Interdigitating foot processes from podocytes form slit diaphragms which regulate the filtration of molecules through size and charge selectivity. The abundance of lipid rafts, which are ordered membrane domains rich in cholesterol and sphingolipids, near the slit diaphragm highlights the importance of lipid metabolism in podocyte health. Emerging research shows the importance of sphingolipid metabolism to podocyte health through structural and signaling roles. Dysregulation in sphingolipid metabolism has been shown to cause podocyte injury and drive glomerular disease progression. In this review, we discuss the structure and metabolism of sphingolipids, as well as their role in proper podocyte function and how alterations in sphingolipid metabolism contributes to podocyte injury and drives glomerular disease progression. [ABSTRACT FROM AUTHOR]

Excessive lipid deposition affects hepatic homeostasis and contributes to the development of insulin resistance as a crucial factor for the deterioration of simple steatosis to steatohepatitis. So, it is essential to search for an effective agent for a new therapy for hepatic steatosis development before it progresses to the more advanced stages. Our study aimed to evaluate the potential protective effect of α-lipoic acid (α-LA) administration on the intrahepatic metabolism of sphingolipid and insulin signaling transduction in rats with metabolic dysfunction-associated steatotic liver disease (MASLD). The experiment was conducted on male Wistar rats subjected to a standard diet or a high-fat diet (HFD) and an intragastrically α-LA administration for eight weeks. High-performance liquid chromatography (HPLC) was used to determine sphingolipid content. Immunoblotting was used to measure the expression of selected proteins from sphingolipid and insulin signaling pathways. Multiplex assay kit was used to assess the level of the phosphorylated form of proteins from PI3K/Akt/mTOR transduction. The results revealed that α-LA decreased sphinganine, dihydroceramide, and sphingosine levels and increased ceramide level. We also observed an increased the concentration of phosphorylated forms of sphingosine and sphinganine. Changes in the expression of proteins from sphingolipid metabolism were consistent with changes in sphingolipid pools. Treatment with α-LA activated the PI3K/Akt/mTOR pathway, which enhanced the hepatic phosphorylation of Akt and mTOR. Based on these data, we concluded that α-lipoic acid may alleviate glucose intolerance and may have a protective influence on the sphingolipid metabolism under HFD; thus, this antioxidant appears to protect from MASLD development and steatosis deterioration. [ABSTRACT FROM AUTHOR]

Simple Summary: Neuroblastoma is the most common cancer in infants and the most common solid tumor outside the brain in children, and responds poorly to current therapies. The sphingolipid-modifying drug opaganib, which has anticancer and anti-inflammatory activity in many preclinical models, was tested for inhibition of neuroblastoma cell proliferation in cell culture and in tumors growing in mice. Opaganib inhibited cell proliferation regardless of the MYCN gene status of the neuroblastoma cells. Treatment of tumor-bearing mice with opaganib in combination with the established drugs irinotecan and temozolomide reduced tumor growth and increased survival compared with irinotecan plus temozolomide alone. Because opaganib has already demonstrated safety in patients with cancer, this new drug may provide improved therapy for neuroblastoma patients. Neuroblastoma (NB), the most common cancer in infants and the most common solid tumor outside the brain in children, grows aggressively and responds poorly to current therapies. We have identified a new drug (opaganib, also known as ABC294640) that modulates sphingolipid metabolism by inhibiting the synthesis of sphingosine 1-phosphate (S1P) by sphingosine kinase-2 and elevating dihydroceramides by inhibition of dihydroceramide desaturase. The present studies sought to determine the potential therapeutic activity of opaganib in cell culture and xenograft models of NB. Cytotoxicity assays demonstrated that NB cells, including cells with amplified MYCN, are effectively killed by opaganib concentrations well below those that accumulate in tumors in vivo. Opaganib was shown to cause dose-dependent decreases in S1P and hexosylceramide levels in Neuro-2a cells, while concurrently elevating levels of dihydroceramides. As with other tumor cells, opaganib reduced c-Myc and Mcl-1 protein levels in Neuro-2a cells, and also reduced the expression of the N-Myc protein. The in vivo growth of xenografts of human SK-N-(BE)2 cells with amplified MYCN was suppressed by oral administration of opaganib at doses that are well tolerated in mice. Combining opaganib with temozolomide plus irinotecan, considered the backbone for therapy of relapsed or refractory NB, resulted in increased antitumor activity in vivo compared with temozolomide plus irinotecan or opaganib alone. Mice did not lose additional weight when opaganib was combined with temozolomide plus irinotecan, indicating that the combination is well tolerated. Opaganib has additive antitumor activity toward Neuro-2a tumors when combined with the checkpoint inhibitor anti-CTLA-4 antibody; however, the combination of opaganib with anti-PD-1 or anti-PD-L1 antibodies did not provide increased antitumor activity over that seen with opaganib alone. Overall, the data demonstrate that opaganib modulates sphingolipid metabolism and intracellular signaling in NB cells and inhibits NB tumor growth alone and in combination with other anticancer drugs. Amplified MYCN does not confer resistance to opaganib, and, in fact, the drug attenuates the expression of both c-Myc and N-Myc. The safety of opaganib has been established in clinical trials with adults with advanced cancer or severe COVID-19, and so opaganib has excellent potential for treating patients with NB, particularly in combination with temozolomide and irinotecan or anti-CTLA-4 antibody. [ABSTRACT FROM AUTHOR]

Zinc oxide nanoparticles (ZnO NPs) are widely used in versatile applications, from high technology to household products. While numerous studies have examined the toxic gene profile of ZnO NPs across various tissues, the specific lipid species associated with adverse effects and potential biomarkers remain elusive. In this study, we conducted a liquid chromatography-mass spectrometry based lipidomics analysis to uncover potential lipid biomarkers in human kidney cells following treatment with ZnO NPs. Furthermore, we employed lipid pathway enrichment analysis (LIPEA) to elucidate altered lipid-related signaling pathways. Our results demonstrate that ZnO NPs induce cytotoxicity in renal epithelial cells and modulate lipid species; we identified 64 lipids with a fold change (FC) > 2 and p < 0.01 with corrected p < 0.05 in HK2 cells post-treatment with ZnO NPs. Notably, the altered lipids between control HK2 cells and those treated with ZnO NPs were associated with the sphingolipid, autophagy, and glycerophospholipid pathways. This study unveils novel potential lipid biomarkers of ZnO NP nanotoxicity, representing the first lipidomic profiling of ZnO NPs in human renal epithelial cells. [ABSTRACT FROM AUTHOR]

Myriocin is an inhibitor of serine palmitoyltransferase involved in the initial biosynthetic step for sphingolipids, and causes potent growth inhibition in eukaryotic cells. In budding yeast, Rsb1, Rta1, Pug1, and Ylr046c are known as the Lipid-Translocating Exporter (LTE) family and believed to contribute to export of various cytotoxic lipophilic compounds. It was reported that Rsb1 is a transporter responsible for export of intracellularly accumulated long-chain bases, which alleviate the cytotoxicity. In this study, it was found that LTE family genes are involved in determination of myriocin sensitivity in yeast. Analyses of effects of deletion and overexpression of LTE family genes suggested that all LTEs contribute to suppression of cytotoxicity of myriocin. It was confirmed that RSB1 overexpression suppressed reduction in complex sphingolipid levels caused by myriocin treatment, possibly exporting myriocin to outside of the cell. These results suggested that LTE family genes function as a defense mechanism against myriocin.

Biological membranes consist of a lipid bilayer in which integral membrane proteins are embedded. Based on the compositional complexity of the lipid species found in membranes, and on their specific and selective interactions with membrane proteins, we recently suggested that membrane bilayers can be best described as “finely-tuned molecular machines.” We now discuss one such set of lipid-protein interactions by describing a negative feedback mechanism operating in the de novo sphingolipid biosynthetic pathway, which occurs in the membrane of the endoplasmic reticulum, and describe the atomic interactions between the first enzyme in the pathway, namely serine palmitoyl transferase, and the product of the fourth enzyme in the pathway, ceramide. We explore how hydrogen-bonding and hydrophobic interactions formed between Asn13 and Phe63 in the serine palmitoyl transferase complex and ceramide can influence the ceramide content of the endoplasmic reticulum. This example of finely-tuned biochemical interactions raises intriguing mechanistic questions about how sphingolipids and their biosynthetic enzymes could have evolved, particularly in light of their metabolic co-dependence.

Abstract Mass spectrometry-based lipidomics approaches offer valuable tools for the detection and quantification of various lipid species, including sphingolipids. The present study aimed to develop a new method to simultaneously detect various sphingolipid species that applies to diverse biological samples. We developed and validated a measurement system by employing a single-column liquid chromatography-mass spectrometry system utilizing a normal-phase separation mode with positive ionization. The measurement system provided precision with a coefficient of variant below 20% for sphingolipids in all types of samples, and we observed good linearity in diluted serum samples. This system can measure the following sphingolipids: sphingosine 1-phosphate (S1P), sphingosine (Sph), dihydroS1P (dhS1P), dihydroSph (dhSph), ceramide 1-phosphate (Cer1P), hexosylceramide (HexCer), lactosylceramide (LacCer), dh-ceramide, deoxy-ceramide, deoxy-dh-ceramide, and sphingomyelin (SM). By measuring these sphingolipids in cell lysates where S1P lyase expression level was modulated, we could observe significant and dynamic modulations of sphingolipids in a comprehensive manner. Our newly established and validated measurement system can simultaneously measure many kinds of sphingolipids in biological samples. It holds great promise as a valuable tool for laboratory testing applications to detect overall modulations of sphingolipids, which have been proposed to be involved in pathogenesis processes in a series of elegant basic research studies.

The quality of strong-flavor Baijiu, a prominent Chinese liquor, is intricately tied to the choice of sorghum variety used in fermentation. However, a significant gap remains in our understanding of how glutinous and non-glutinous sorghum varieties comprehensively impact Baijiu flavor formation through fermentation metabolites. This study employed untargeted metabolomics combined with feature-based molecular networking (FBMN) to explore the unique metabolic characteristics of these two sorghum varieties during fermentation. FBMN analysis revealed 267 metabolites within both types of fermented sorghum (Zaopei) in the cellar. Further multidimensional statistical analyses highlighted sphingolipids, 2,5-diketopiperazines, and methionine derivatives as critical markers for quality control. These findings represent a significant advancement in our understanding and provide valuable insights for regulating the quality of Baijiu flavors.

The bioactive sphingolipid ceramide impacts diverse cellular processes (e.g. apoptosis and cell proliferation) through its effects on membrane dynamics and intracellular signaling pathways. The dysregulation of ceramide metabolism has been implicated in cancer evasion of apoptosis and targeting ceramide metabolism has potential therapeutic benefits as a strategy to kill cancer cells and slow tumor growth. However, the mechanisms of cancer cell resistance to ceramide-mediated cell death are vastly intertwined and incompletely understood. To shed light on this mystery, we performed a genome-wide CRISPR-Cas9 screen to systematically identify regulators of cancer resistance to the soluble short chain ceramide, C6 ceramide (C6-Cer). Our results reveal a complex landscape of genetic modifiers of C6-Cer toxicity, including genes associated with ceramide and sphingolipid metabolism, vesicular trafficking, and membrane biology. Furthermore, we find that loss of the phospholipid flippase subunit TMEM30A impairs the plasma membrane trafficking of its binding partner, the P4-type ATPase ATP11B, and depletion of TMEM30A or ATP11B disrupts plasma membrane asymmetry and promotes resistance to C6-Cer toxicity. Together, our findings provide a resource of genetic modifiers of C6-Cer toxicity and reveal an unexpected role of plasma membrane asymmetry in C6-Cer induced cell death.

Pain is a clinical condition that is currently of great concern and is often caused by tissue or nerve damage or occurs as a concomitant symptom of a variety of diseases such as cancer. Severe pain seriously affects the functional status of the body. However, existing pain management programs are not fully satisfactory. Therefore, there is a need to delve deeper into the pathological mechanisms underlying pain generation and to find new targets for drug therapy. Sphingolipids (SLs), as a major component of the bilayer structure of eukaryotic cell membranes, also have powerful signal transduction functions. Sphingolipids are abundant, and their intracellular metabolism constitutes a huge network. Sphingolipids and their various metabolites play significant roles in cell proliferation, differentiation, apoptosis, etc., and have powerful biological activities. The molecules related to sphingolipid metabolism, mainly the core molecule ceramide and the downstream metabolism molecule sphingosine-1-phosphate (S1P), are involved in the specific mechanisms of neurological disorders as well as the onset and progression of various types of pain, and are closely related to a variety of pain-related diseases. Therefore, sphingolipid metabolism can be the focus of research on pain regulation and provide new drug targets and ideas for pain. [ABSTRACT FROM AUTHOR]

Brain health requires circuits, cells and molecular pathways to adapt when challenged and to promptly reset once the challenge has resolved. Neurodegeneration occurs when adaptability becomes confined, causing challenges to overwhelm neural circuitry. Studies of rare and common neurodegenerative diseases suggest that the accumulation of lipids can compromise circuit adaptability. Using microglia as an example, we review data that suggest increased lipid concentrations cause dysfunctional inflammatory responses to immune challenges, leading to Alzheimer's disease, Parkinson's disease and dementia. We highlight current approaches to treat lipid metabolic and clearance pathways and identify knowledge gaps towards restoring adaptive homeostasis in individuals who are at‐risk of losing cognition. [ABSTRACT FROM AUTHOR]

Fibrosis is a chronic pathology resulting from excessive deposition of extracellular matrix components that leads to the loss of tissue function. Pulmonary fibrosis can follow a variety of diverse insults including ischemia, respiratory infection, or exposure to ionizing radiation. Consequently, treatments that attenuate the development of debilitating fibrosis are in desperate need across a range of conditions. Sphingolipid metabolism is a critical regulator of cell proliferation, apoptosis, autophagy, and pathologic inflammation, processes that are all involved in fibrosis. Opaganib (formerly ABC294640) is the first-in-class investigational drug targeting sphingolipid metabolism for the treatment of cancer and inflammatory diseases. Opaganib inhibits key enzymes in sphingolipid metabolism, including sphingosine kinase-2 and dihydroceramide desaturase, thereby reducing inflammation and promoting autophagy. Herein, we demonstrate in mouse models of lung damage following exposure to ionizing radiation that opaganib significantly improved long-term survival associated with reduced lung fibrosis, suppression of granulocyte infiltration, and reduced expression of IL-6 and TNFα at 180 days after radiation. These data further demonstrate that sphingolipid metabolism is a critical regulator of fibrogenesis, and specifically show that opaganib suppresses radiation-induced pulmonary inflammation and fibrosis. Because opaganib has demonstrated an excellent safety profile during clinical testing in other diseases (cancer and COVID-19), the present studies support additional clinical trials with this drug in patients at risk for pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Background: Oral squamous cell carcinoma has high recurrence and cisplatin resistance. As cancer stem cells, autophagy, and sphingolipids have been appointed as associated with chemotherapy resistance, we tested combined treatments targeting autophagy and/or sphingolipid metabolism with paclitaxel using cisplatin‐resistant oral squamous cell carcinoma cells. Methods: Cisplatin‐resistant oral squamous cell carcinoma cells were maintained under exposition to FTY720 and chloroquine combined with paclitaxel and submitted to viability, clonogenicity, and spheres formation assays. The xenograft tumor model using cisplatin‐resistant CAL27 cells was adopted to examine the drug combinations' potential antitumoral efficacy. Using an animal model, sphingolipids profiles from plasma and tissue samples were obtained by liquid chromatography coupled to mass spectrometry to identify potential lipids associated with drug response. Results and Discussion: Our results showed higher autophagic flux in cisplatin‐resistant Ooral squamous cell carcinoma (CAL27 and SCC9) cells than in parental cells. The combinations of an autophagy inhibitor (chloroquine) or an autophagy inducer/sphingosine kinase 1 antagonist (FTY720) with paclitaxel (PTX) had a synergistic antitumor effect. Treated CisR cells lost clonogenicity and tumor sphere abilities and reduced proteins associated with proliferation, survival, and cancer stem cells. FTY720 plus PTX had higher antitumor efficacy than PTX against CAL27 CisR xenograft tumor formation. Additionally, increases in glucosylceramide, dehydroglucosylceramide, and sphingomyelin were presented in responsive tumors. Conclusion: FTY720 sensitizes cisplatin‐resistant oral squamous cell carcinoma cells for paclitaxel. [ABSTRACT FROM AUTHOR]

Abnormalities of sphingolipid metabolism play an important role in diabetes. We compared sphingolipid levels in plasma and in isolated lipoproteins between healthy control subjects and two groups of patients, one with chronic kidney disease without diabetes (ND-CKD), and the other with type 2 diabetes and macroalbuminuria (D-MA). Ceramides, sphingomyelins, and sphingoid bases and their phosphates in LDL were higher in ND-CKD and in D-MA patients compared to controls. However, ceramides and sphingoid bases in HDL2 and HDL3 were lower in ND-CKD and in D-MA patients than in controls. Sphingomyelins in HDL2 and HDL3 were lower in D-MA patients than in controls but were normal in ND-CKD patients. Compared to controls, lactosylceramides in LDL and VLDL were higher in ND-CKD patients but not in D-MA patients. However, lactosylceramides in HDL2 and HDL3 were lower in both ND-CKD and D-MA patients than in controls. Plasma hexosylceramides in ND-CKD patients were increased and sphingoid bases decreased in both ND-CKD and D-MA patients. However, hexosylceramides in LDL, HDL2, and HDL3 were higher in ND-CKD patients than in controls. In D-MA patients, only C16:0 hexosylceramide in LDL was higher than in controls. The data suggest that sphingolipid measurement in lipoproteins, rather than in whole plasma, is crucial to decipher the role of sphingolipids in kidney disease. [ABSTRACT FROM AUTHOR]

Multiple isozymes are encoded in the Caenorhabditis elegans genome for the various sphingolipid biosynthesis reactions, but the contributions of individual isozymes are characterized only in part. We developed a simple but effective reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method that enables simultaneous identification and quantification of ceramides (Cer), glucosylceramides (GlcCer), and sphingomyelins (SM) from the same MS run. Validating this sphingolipid profiling method, we show that nearly all 47 quantifiable sphingolipid species found in young adult worms were reduced upon RNA interference (RNAi) of sptl-1 or elo-5, which are both required for synthesis of the id17:1 sphingoid base. We also confirm that HYL-1 and HYL-2, but not LAGR-1, constitute the major ceramide synthase activity with different preference for fatty acid substrates, and that CGT-3, but not CGT-1 and CGT-2, plays a major role in producing GlcCers. Deletion of sms-5 hardly affected SM levels. RNAi of sms-1, sms-2, and sms-3 all lowered the abundance of certain SMs with an odd-numbered N-acyl chains (mostly C21 and C23, with or without hydroxylation). Unexpectedly, sms-2 RNAi and sms-3 RNAi elevated a subset of SM species containing even-numbered N-acyls. This suggests that sphingolipids containing even-numbered N-acyls could be regulated separately, sometimes in opposite directions, from those containing odd-numbered N-acyls, which are presumably monomethyl branched chain fatty acyls. We also find that ceramide levels are kept in balance with those of GlcCers and SMs. These findings underscore the effectiveness of this RPLC-MS/MS method in studies of C. elegans sphingolipid biology.

Adipogenesis is one of the major mechanisms for adipose tissue expansion, during which spindle-shaped mesenchymal stem cells commit to the fate of adipocyte precursors and differentiate into round-shaped fat-laden adipocytes. Here, we investigated the lipidomic profile dynamics of ex vivo-differentiated brown and white adipocytes derived from the stromal vascular fractions of interscapular brown (iBAT) and inguinal white adipose tissues. We showed that sphingomyelin was specifically enriched in terminally differentiated brown adipocytes, but not white adipocytes. In line with this, freshly isolated adipocytes of iBAT showed higher sphingomyelin content than those of inguinal white adipose tissue. Upon cold exposure, sphingomyelin abundance in iBAT gradually decreased in parallel with reduced sphingomyelin synthase 1 protein levels. Cold-exposed animals treated with an inhibitor of sphingomyelin hydrolases failed to maintain core body temperature and showed reduced oxygen consumption and iBAT UCP1 levels. Conversely, blockade of sphingomyelin synthetic enzymes resulted in enhanced nonshivering thermogenesis, reflected by elevated body temperature and UCP1 levels. Taken together, our results uncovered a relation between sphingomyelin abundance and fine-tuning of UCP1-mediated nonshivering thermogenesis.

The synapse is a piece of information transfer machinery replacing the electrical conduction of nerve impulses at the end of the neuron. Like many biological mechanisms, its functioning is heavily affected by time constraints. The solution selected by evolution is based on chemical communication that, in theory, cannot compete with the speed of nerve conduction. Nevertheless, biochemical and biophysical compensation mechanisms mitigate this intrinsic weakness: (i) through the high concentrations of neurotransmitters inside the synaptic vesicles; (ii) through the concentration of neurotransmitter receptors in lipid rafts, which are signaling platforms; indeed, the presence of raft lipids, such as gangliosides and cholesterol, allows a fine tuning of synaptic receptors by these lipids; (iii) through the negative electrical charges of the gangliosides, which generate an attractive (for cationic neurotransmitters, such as serotonin) or repulsive (for anionic neurotransmitters, such as glutamate) electric field. This electric field controls the flow of glutamate in the tripartite synapse involving pre- and post-synaptic neurons and the astrocyte. Changes in the expression of brain gangliosides can disrupt the functioning of the glutamatergic synapse, causing fatal diseases, such as Rett syndrome. In this review, we propose an in-depth analysis of the role of gangliosides in the glutamatergic synapse, highlighting the primordial and generally overlooked role played by the electric field of synaptic gangliosides.

The (patho)physiological function of the sphingolipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), and sphingosylphosphorylcholine (SPC) in articular joints during osteoarthritis (OA) is largely unknown. Therefore, we investigated the influence of these lipids on protein expression by fibroblast-like synoviocytes (FLSs) from OA knees. Cultured human FLSs (n = 7) were treated with 1 of 3 lipid species—C1P, S1P, or SPC—IL-1β, or with vehicle. The expression of individual proteins was determined by tandem mass tag peptide labeling followed by high-resolution electrospray ionization (ESI) mass spectrometry after liquid chromatographic separation (LC-MS/MS/MS). The mRNA levels of selected proteins were analyzed using RT-PCR. The 3sphingolipids were quantified in the SF of 18 OA patients using LC-MS/MS. A total of 4930 proteins were determined using multiplex MS, of which 136, 9, 1, and 0 were regulated both reproducibly and significantly by IL-1β, C1P, S1P, and SPC, respectively. In the presence of IL-1ß, all 3 sphingolipids exerted ancillary effects. Only low SF levels of C1P and SPC were found. In conclusion, the 3 lipid species regulated proteins that have not been described in OA. Our results indicate that charged multivesicular body protein 1b, metal cation symporter ZIP14, glutamine-fructose-6-P transaminase, metallothionein-1F and -2A, ferritin, and prosaposin are particularly interesting proteins due to their potential to affect inflammatory, anabolic, catabolic, and apoptotic mechanisms.

Lipid A, the membrane-anchored portion of lipopolysaccharide (LPS), is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here we identify regulatory and structural factors that together render lipid A nonessential in Caulobacter crescentus. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to survive when lipid A synthesis is blocked. Fitness of lipid A-deficient Caulobacter requires an anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.

Background: We explore sphingolipid-related genes (SRGs) in skin melanoma (SKCM) to develop a prognostic indicator for patient outcomes. Dysregulated lipid metabolism is linked to aggressive behavior in various cancers, including SKCM. However, the exact role and mechanism of sphingolipid metabolism in melanoma remain partially understood. Methods: We integrated scRNA-seq data from melanoma patients sourced from the GEO database. Through the utilization of the Seurat R package, we successfully identified distinct gene clusters associated with patient survival in the scRNA-seq data. Key prognostic genes were identified through single-factor Cox analysis and used to develop a prognostic model using LASSO and stepwise regression algorithms. Additionally, we evaluated the predictive potential of these genes within the immune microenvironment and their relevance to immunotherapy. Finally, we validated the functional significance of the highrisk gene IRX3 through in vitro experiments. Results: Analysis of scRNA-seq data identified distinct expression patterns of 4 specific genes (SRGs) in diverse cell subpopulations. Re-clustering cells based on increased SRG expression revealed 7 subgroups with significant prognostic implications. Using marker genes, lasso, and Cox regression, we selected 11 genes to construct a risk signature. This signature demonstrated a strong correlation with immune cell infiltration and stromal scores, highlighting its relevance in the tumor microenvironment. Functional studies involving IRX3 knockdown in A375 and WM-115 cells showed significant reductions in cell viability, proliferation, and invasiveness. Conclusion: SRG-based risk signature holds promise for precise melanoma prognosis. An in-depth exploration of SRG characteristics offers insights into immunotherapy response. Therapeutic targeting of the IRX3 gene may benefit melanoma patients. [ABSTRACT FROM AUTHOR]

The bioactive sphingolipid sphingosine-1-phosphate (S1P) acts as a ligand for a family of G protein-coupled S1P receptors (S1PR1-5) to participate in a variety of signaling pathways. However, their specific roles in the neural retina remain unclear. We previously showed that S1P receptor subtype 2 (S1PR2) is expressed in murine retinas, primarily in photoreceptors and bipolar cells, and its expression is altered by retinal stress. This study aims to elucidate the role of S1PR2 in the mouse retina. We examined light responses by electroretinography (ERG), structural differences by optical coherence tomography (OCT), and protein levels by immunohistochemistry (IHC) in wild-type (WT) and S1PR2 knockout (KO) mice at various ages between 3 and 6 months. We found that a- and b-wave responses significantly increased at flash intensities between 400~2000 and 4~2000 cd.s/m2, respectively, in S1PR2 KO mice relative to those of WT controls at baseline. S1PR2 KO mice also exhibited significantly increased retinal nerve fiber layer (RNFL) and outer plexiform layer (OPL) thickness by OCT relative to the WT. Finally, in S1PR2 KO mice, we observed differential labeling of synaptic markers by immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (RT-qPCR). These results suggest a specific involvement of S1PR2 in the structure and synaptic organization of the retina and a potential role in light-mediated functioning of the retina. [ABSTRACT FROM AUTHOR]

Great strides in the field of lipidomics driven by advances in mass spectrometry techniques in the last decade have moved lipid analysis to a new level and significantly improved our understanding of lipid biochemistry. Multiple stage mass spectrometry (MSn) with high resolution mass spectrometry (HRMS) that allows sequential isolation, fragmentation, and recognition of ion structures, is a powerful tool for characterization of complex and diversified lipid in bacterial cells, in which lipids are often critical for cell aggregation and dissociation, and play important biological roles. In addition to common phospholipids, many bacteria contain unique lipids that are specific to the bacterium genus and even to the bacterium species. In this review, application of linear ion-trap (LIT) MSn in the structural characterization of native bacterial lipids including (1) novel lipids consisting of many isomeric structures, (2) lipids with unique functional groups and modification, (3) complex sphingolipids, peptidolipids, and lipocyclopeptides from various bacteria are presented. LIT MSn approach affords realization of the mechanisms underlying the fragmentation processes, resulting in identification of complex lipid structures that would be very difficult to define using other analytical methods. • LIT MSn with high resolution mass spectrometric approach towards characterization of bacterial lipids is described. • Uncommon bacterial lipids containing many isomers, unique functional groups are reviewed. • LIT MSn affords revelation of fragmentation mechanisms, leading to complete structural identification. One sentence summary: This review gives an overview on mass spectrometric approach toward structural definition of the diversified bacterial membrane lipids. [ABSTRACT FROM AUTHOR]

The basic structures of membrane lipids that compose biomembranes differ among species; i.e., in mammals, the primary structure of long‐chain base (LCB), the common backbone of ceramides and complex sphingolipids, is sphingosine, whereas, in yeast Saccharomyces cerevisiae, it is phytosphingosine, and S. cerevisiae does not have sphingosine. In addition, the sterol, which is coordinately involved in various functions with complex sphingolipids, is cholesterol in mammals, while in yeast it is ergosterol. Previously, it was found that yeast cells are viable when the structure of LCBs is replaced by sphingosine by supplying an exogenous LCB to cells lacking LCB biosynthesis. Here, we characterized yeast cells having sphingosine instead of phytosphingosine (sphingosine cells). Sphingosine cells exhibited a strong growth defect when biosynthesis of ceramides or complex sphingolipids was inhibited, indicating that, in the sphingosine cells, exogenously added sphingosine is required to be further metabolized. The sphingosine cells exhibited hypersensitivity to various environmental stresses and had abnormal plasma membrane and cell wall properties. Furthermore, we also established a method for simultaneous replacement of both LCB and sterol structures with those of mammals (sphingosine/cholesterol cells). The multiple stress hypersensitivity and abnormal plasma membrane and cell wall properties observed in sphingosine cells were also observed in sphingosine/cholesterol cells, suggesting that simultaneous replacement of both LCB and sterol structures with those of mammals cannot prevent these abnormal phenotypes. This is the first study to our knowledge showing that S. cerevisiae can grow even if LCB and sterol structures are simultaneously replaced with mammalian types. [ABSTRACT FROM AUTHOR]

Antibody-based cancer drugs that target the checkpoint proteins CTLA-4, PD-1 and PD-L1 provide marked improvement in some patients with deadly diseases such as lung cancer and melanoma. However, most patients are either unresponsive or relapse following an initial response, underscoring the need for further improvement in immunotherapy. Certain drugs induce immunogenic cell death (ICD) in tumor cells in which the dying cells promote immunologic responses in the host that may enhance the in vivo activity of checkpoint antibodies. Sphingolipid metabolism is a key pathway in cancer biology, in which ceramides and sphingosine 1-phosphate (S1P) regulate tumor cell death, proliferation and drug resistance, as well as host inflammation and immunity. In particular, sphingosine kinases are key sites for manipulation of the ceramide/S1P balance that regulates tumor cell proliferation and sensitivity to radiation and chemotherapy. We and others have demonstrated that inhibition of sphingosine kinase-2 by the small-molecule investigational drug opaganib (formerly ABC294640) kills tumor cells and increases their sensitivities to other drugs and radiation. Because sphingolipids have been shown to regulate ICD, opaganib may induce ICD and improve the efficacy of checkpoint antibodies for cancer therapy. This was demonstrated by showing that in vitro treatment with opaganib increases the surface expression of the ICD marker calreticulin on a variety of tumor cell types. In vivo confirmation was achieved using the gold standard immunization assay in which B16 melanoma, Lewis lung carcinoma (LLC) or Neuro-2a neuroblastoma cells were treated with opaganib in vitro and then injected subcutaneously into syngeneic mice, followed by implantation of untreated tumor cells 7 days later. In all cases, immunization with opaganib-treated cells strongly suppressed the growth of subsequently injected tumor cells. Interestingly, opaganib treatment induced crossover immunity in that opaganib-treated B16 cells suppressed the growth of both untreated B16 and LLC cells and opaganib-treated LLC cells inhibited the growth of both untreated LLC and B16 cells. Next, the effects of opaganib in combination with a checkpoint antibody on tumor growth in vivo were assessed. Opaganib and anti-PD-1 antibody each slowed the growth of B16 tumors and improved mouse survival, while the combination of opaganib plus anti-PD-1 strongly suppressed tumor growth and improved survival (p < 0.0001). Individually, opaganib and anti-CTLA-4 antibody had modest effects on the growth of LLC tumors and mouse survival, whereas the combination of opaganib with anti-CTLA-4 substantially inhibited tumor growth and increased survival (p < 0.001). Finally, the survival of mice bearing B16 tumors was only marginally improved by opaganib or anti-PD-L1 antibody alone but was nearly doubled by the drugs in combination (p < 0.005). Overall, these studies demonstrate the ability of opaganib to induce ICD in tumor cells, which improves the antitumor activity of checkpoint antibodies. [ABSTRACT FROM AUTHOR]

Sleep is a fundamental state of behavioral quiescence and physiological restoration. Sleep is controlled by environmental conditions, indicating a complex regulation of sleep by multiple processes. Our knowledge of the genes and mechanisms that control sleep during various conditions is, however, still incomplete. In Caenorhabditis elegans, sleep is increased when development is arrested upon starvation. Here, we performed a reverse genetic sleep screen in arrested L1 larvae for genes that are associated with metabolism. We found over 100 genes that are associated with a reduced sleep phenotype. Enrichment analysis revealed sphingolipid metabolism as a key pathway that controls sleep. A strong sleep loss was caused by the loss of function of the diacylglycerol kinase 1 gene, dgk-1, a negative regulator of synaptic transmission. Rescue experiments indicated that dgk-1 is required for sleep in cholinergic and tyraminergic neurons. The Ring Interneuron S (RIS) neuron is crucial for sleep in C. elegans and activates to induce sleep. RIS activation transients were abolished in dgk-1 mutant animals. Calcium transients were partially rescued by a reduction-of-function mutation of unc-13, suggesting that dgk-1 might be required for RIS activation by limiting synaptic vesicle release. dgk-1 mutant animals had impaired L1 arrest survival and dampened expression of the protective heat shock factor gene hsp-12.6. These data suggest that dgk-1 impairment causes broad physiological deficits. Microcalorimetry and metabolomic analyses of larvae with impaired RIS showed that RIS is broadly required for energy conservation and metabolic control, including for the presence of sphingolipids. Our data support the notion that metabolism broadly influences sleep and that sleep is associated with profound metabolic changes. We thus provide novel insights into the interplay of lipids and sleep and provide a rich resource of mutants and metabolic pathways for future sleep studies. [ABSTRACT FROM AUTHOR]

The dynamic balance and distribution of sphingolipid metabolites modulate the level of programmed cell death and plant defence. However, current knowledge is still limited regarding the molecular mechanism underlying the relationship between sphingolipid metabolism and plant defence. In this study, we identified a wheat RNA‐binding protein 1 (TaRBP1) and TaRBP1 mRNA accumulation significantly decreased in wheat after infection by Puccinia striiformis f. sp. tritici (Pst). Knockdown of TaRBP1 via virus‐induced gene silencing conferred strong resistance to Pst by enhancing host plant reactive oxygen species (ROS) accumulation and cell death, indicating that TaRBP1 may act as a negative regulator in response to Pst. TaRBP1 formed a homopolymer and interacted with TaRBP1 C‐terminus in plants. Additionally, TaRBP1 physically interacted with TaGLTP, a sphingosine transfer protein. Knockdown of TaGLTP enhanced wheat resistance to the virulent Pst CYR31. Sphingolipid metabolites showed a significant accumulation in TaGLTP‐silenced wheat and TaRBP1‐silenced wheat, respectively. In the presence of the TaRBP1 protein, TaGLTP failed to be degraded in a 26S proteasome‐dependent manner in plants. Our results reveal a novel susceptible mechanism by which a plant fine‐tunes its defence responses by stabilizing TaGLTP accumulation to suppress ROS and sphingolipid accumulation during Pst infection. [ABSTRACT FROM AUTHOR]

Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here, we report that deletion of tricalbins (Tcb1, Tcb2, and Tcb3), tethering proteins at endoplasmic reticulum (ER)–plasma membrane (PM) and ER–Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine (PHS) accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus–vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous PHS and Tcb3-deleted cells, supporting that PHS transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.

Age-related muscle wasting and dysfunction render the elderly population vulnerable and incapacitated, while underlying mechanisms are poorly understood. Here, we implicate the CERS1 enzyme of the de novo sphingolipid synthesis pathway in the pathogenesis of age-related skeletal muscle impairment. In humans, CERS1 abundance declines with aging in skeletal muscle cells and, correlates with biological pathways involved in muscle function and myogenesis. Furthermore, CERS1 is upregulated during myogenic differentiation. Pharmacological or genetic inhibition of CERS1 in aged mice blunts myogenesis and deteriorates aged skeletal muscle mass and function, which is associated with the occurrence of morphological features typical of inflammation and fibrosis. Ablation of the CERS1 orthologue lagr-1 in Caenorhabditis elegans similarly exacerbates the age-associated decline in muscle function and integrity. We discover genetic variants reducing CERS1 expression in human skeletal muscle and Mendelian randomization analysis in the UK biobank cohort shows that these variants reduce muscle grip strength and overall health. In summary, our findings link age-related impairments in muscle function to a reduction in CERS1, thereby underlining the importance of the sphingolipid biosynthesis pathway in age-related muscle homeostasis.

ABSTRACT Sphingofungins are sphinganine analog mycotoxins acting as inhibitors of serine palmitoyl transferases, enzymes responsible for the first step in the sphingolipid biosynthesis. Eukaryotic cells are highly organized with various structures and organelles to facilitate cellular processes and chemical reactions, including the ones occurring as part of the secondary metabolism. We studied how sphingofungin biosynthesis is compartmentalized in the human-pathogenic fungus Aspergillus fumigatus, and we observed that it takes place in the endoplasmic reticulum (ER), ER-derived vesicles, and the cytosol. This implies that sphingofungin and sphingolipid biosynthesis colocalize to some extent. Automated analysis of confocal microscopy images confirmed the colocalization of the fluorescent proteins. Moreover, we demonstrated that the cluster-associated aminotransferase (SphA) and 3-ketoreductase (SphF) play a bifunctional role, supporting sphingolipid biosynthesis, and thereby antagonizing the toxic effects caused by sphingofungin production.IMPORTANCEA balanced sphingolipid homeostasis is critical for the proper functioning of eukaryotic cells. To this end, sphingolipid inhibitors have therapeutic potential against diseases related to the deregulation of sphingolipid balance. In addition, some of them have significant antifungal activity, suggesting that sphingolipid inhibitors-producing fungi have evolved mechanisms to escape self-poisoning. Here, we propose a novel self-defense mechanism, with cluster-associated genes coding for enzymes that play a dual role, being involved in both sphingofungin and sphingolipid production.

Lipid transport is an essential cellular process with importance to human health, disease development, and therapeutic strategies. Type IV P-type ATPases (P4-ATPases) have been identified as membrane lipid flippases by utilizing nitrobenzoxadiazole (NBD)-labeled lipids as substrates. Among the 14 human type IV P-type ATPases, ATP10D was shown to flip NBD-glucosylceramide (GlcCer) across the plasma membrane. Here, we found that conversion of incorporated GlcCer (d18:1/12:0) to other sphingolipids is accelerated in cells exogenously expressing ATP10D but not its ATPase-deficient mutant. These findings suggest that 1) ATP10D flips unmodified GlcCer as well as NBD-GlcCer at the plasma membrane and 2) ATP10D can translocate extracellular GlcCer, which is subsequently converted to other metabolites. Notably, exogenous expression of ATP10D led to the reduction in cellular hexosylceramide levels. Moreover, the expression of GlcCer flippases, including ATP10D, also reduced cellular hexosylceramide levels in fibroblasts derived from patients with Gaucher disease, which is a lysosomal storage disorder with excess GlcCer accumulation. Our study highlights the contribution of ATP10D to the regulation of cellular GlcCer levels and maintaining lipid homeostasis.

Abstract Background The risk for dementia increases exponentially from the seventh decade of life. Identifying and understanding the biochemical changes that sensitize the ageing brain to neurodegeneration will provide new opportunities for dementia prevention and treatment. This study aimed to determine how ageing and major genetic risk factors for dementia affect the hippocampal proteome and lipidome of neurologically-normal humans over the age of 65. The hippocampus was chosen as it is highly susceptible to atrophy with ageing and in several neurodegenerative diseases. Methods Mass spectrometry-based proteomic and lipidomic analysis of CA1 hippocampus samples from 74 neurologically normal human donors, aged 66–104, was used in combination with multiple regression models and gene set enrichment analysis to identify age-dependent changes in the proteome and lipidome. ANOVA was used to test the effect of major dementia risk alleles in the TMEM106B and APOE genes on the hippocampal proteome and lipidome, adjusting for age, gender, and post-mortem interval. Fibrillar C-terminal TMEM106B fragments were isolated using sarkosyl fractionation and quantified by immunoblotting. Results Forty proteins were associated with age at false discovery rate-corrected P

Accumulating evidence suggests that various sphingolipids and glycosphingolipids can act as mediators for inflammation or signaling molecules in the nervous system. In this article, we explore the molecular basis of a new neuroinflammatory disorder called encephalomyeloradiculoneuropathy (EMRN), which affects the brain, spinal cord, and peripheral nerves; in particular, we discuss whether glycolipid and sphingolipid dysmetabolism is present in patients with this disorder. This review will focus on the pathognomonic significance of sphingolipid and glycolipid dysmetabolism for the development of EMRN and the possible involvement of inflammation in the nervous system.