Sphingomyelinase Research Articles

Extracellular tau stimulates phagocytosis of living neurons by activated microglia via Toll-like 4 receptor–NLRP3 inflammasome–caspase-1 signalling axis

In tauopathies, abnormal deposition of intracellular tau protein followed by gradual elevation of tau in cerebrospinal fluids and neuronal loss has been documented, however, the mechanism how actually neurons die under tau pathology is largely unknown. We have previously shown that extracellular tau protein (2N4R isoform) can stimulate microglia to phagocytose live neurons, i.e. cause neuronal death by primary phagocytosis, also known as phagoptosis. Here we show that tau protein induced caspase-1 activation in microglial cells via ‘Toll-like’ 4 (TLR4) receptors and neutral sphingomyelinase. Tau-induced neuronal loss was blocked by caspase-1 inhibitors (Ac-YVAD-CHO and VX-765) as well as by TLR4 antibodies. Inhibition of caspase-1 by Ac-YVAD-CHO prevented tau-induced exposure of phosphatidylserine on the outer leaflet of neuronal membranes and reduced microglial phagocytic activity. We also show that suppression of NLRP3 inflammasome, which is down-stream of TLR4 receptors and mediates caspase-1 activation, by a specific inhibitor (MCC550) also prevented tau-induced neuronal loss. Moreover, NADPH oxidase is also involved in tau-induced neurotoxicity since neuronal loss was abolished by its pharmacological inhibitor. Overall, our data indicate that extracellular tau protein stimulates microglia to phagocytose live neurons via Toll-like 4 receptor–NLRP3 inflammasome–caspase-1 axis and NADPH oxidase, each of which may serve as a potential molecular target for pharmacological treatment of tauopathies.

Chronic nSMase inhibition suppresses neuronal exosome spreading and sex-specifically attenuates amyloid pathology in APP knock-in Alzheimer's disease mice

Female biased pathology and cognitive decline in Alzheimer's disease (AD) have been consistently observed with unclear underlying mechanisms. Although brain sphingolipid ceramide is elevated in AD patients, whether and how ceramide may contribute to sex-specific differences in amyloid pathology is unknown. Here we investigated the sex-specific impact of chronic pharmacological inhibition of neutral sphingomyelinase (nSMase), a key enzyme responsible for ceramide metabolism, on in vivo neuron-derived exosome dynamics, Aβ plaque load, and cognitive function in the APPNL-F/NL-F knock-in (APP NL-F) AD mouse model. Our results found sex-specific increase of cortical C20:0 ceramide and brain exosome levels only in APP NL-F but not in age-matched WT mice. Although nSMase inhibition similarly blocks exosome spreading in male and female mice, significantly reduced amyloid pathology was mostly observed in cortex and hippocampus of female APP NL-F mice with only modest effect found on male APP NL-F mice. Consistently, T maze test to examine spatial working memory revealed a female-specific reduction in spontaneous alternation rate in APP NL-F mice, which was fully reversed with chronic nSMase inhibition. Together, our results suggest that disease induced changes in ceramide and exosome pathways contribute to the progression of female-specific amyloid pathology in APP NL-F AD models.

1609-P: Neutral Sphingomyelinase Inhibition Limits Hepatic-Lipotoxicity and Inflammation in a Cell Model of Steatosis

Nonalcoholic fatty liver disease (NAFLD) represents a global healthcare challenge; it is the hepatic manifestation of metabolic syndrome and is strongly associated with developing type 2diabetes mellitus (T2DM). Liver fat accumulation is the first step of disease progression that triggers hepatic lipotoxicity. The role of ceramides in inducing deleterious effects on hepatic metabolism is now well-accepted. Yet, the specific role of stress responsive sphingomyelinase activation under lipotoxic conditions remains under-investigated. The complexity of NAFLD pathogenesis contributes to lack of proper investigatory model, limiting progression in developing and testing novel treatment and prevention strategies. Here, we first report a convenient in-vitro human cell-based model with great resemblance to in-vivo NAFLD hallmarks. Neutral Sphingomyelinase (nSMase2) expression and activity was found to be elevated in both the liver of high fat steatosis mouse models and in HepG2-steatosis cell models. Meanwhile, the functional inhibition of nSMase2 prevented hepatotoxicity-induced pathologies by significantly reducing intracellular lipid accumulation and prevented the upregulation of TNF-α triggered inflammation. Furthermore, inhibition of nSMase2 showed significant increase in PPARα at both gene and protein levels, while PPARα reduction was observed under the stimulation of nSMase2 activity by its agonist daunorubicin (DNR). Together the presented data highlight the role of nSMase2 in the pathogenesis of NAFLD and other disorders linked to hepatic steatosis, providing a novel therapeutic target. Disclosure F. Alrashed: None. H. Arefanian: None. S.T. Sindhu: None. F. Bahman: None. H. AlSaeed: None. A. Al Madhoun: None. F. Alzaid: None. F. Al-Mulla: None. R. Ahmad: None. Funding Kuwait Foundation for the Advancement of Sciences (RA0402021)

1638-P: Oxidized Lipids Cause Ceramide Accumulation and Insulin Resistance in Skeletal Muscle

Accumulation of muscle lipids promotes insulin resistance and type 2 diabetes (T2D). Ceramides are important muscle lipids that decrease insulin sensitivity and it was previously shown that accumulation of ceramides in the sarcolemma negatively impacts insulin sensitivity in humans. The mechanisms explaining accumulation of lipids in specific compartments in skeletal muscle are unknown, but one possibility is through circulating oxidized lipids. POVPC, a major circulating oxidized phospholipid, was exposed to rat myotubes (1-50 μM) for 4h and the total cellular ceramide levels in myotubes measured by LC-MS/MS. Total ceramide content increased dose dependently at POVPC doses in the physiological range (5 μM, p <0.05). To determine the mechanism of POVPC induced ceramide accumulation, ceramide was measured in myotubes after treatment with POVPC (25 μM) in the presence of inhibitors of neutral and acid sphingomyelinase (GW4869 and ARC39), ceramide synthase (fumonisin B1) and serine palmitoyltransferase (myriocin). The individually administered inhibitors in the presence of POVPC each showed a decrease (41-55%) in ceramide accumulation. When all of inhibitors were administered together along with POVPC, an almost complete inhibition of ceramide accumulation (95%) was observed indicating that the sphingomyelinase, de novo, and salvage pathways of ceramide synthesis are activated by POVPC. The role of oxidized lipids on insulin sensitivity was examined after treatment of rat myotubes with 1-25 μM POVPC. Increasing doses of POVPC at doses as low as 1 μM significantly decreased insulin sensitivity in vitro (p <0.05). These results reveal circulating oxidized lipids promote ceramide accumulation and insulin resistance in muscle cells and may help explain variability in total and localized muscle ceramide levels between individuals. These data also suggest that therapies designed to lower circulating oxidized lipids could be beneficial for the treatment and prevention of T2D. Disclosure K.Zemski berry: None. B.C.Bergman: Research Support; Eli Lilly and Company. Funding National Institutes of Health (P30DK048520); University of Colorado (25N0350)

Photosensitive small extracellular vesicles regulate the immune microenvironment of triple negative breast cancer

Currently, the treatment of triple-negative breast cancer (TNBC) is limited by the special pathological characteristics of this disease. In recent years, photodynamic therapy (PDT) has created new hope for the treatment of TNBC. Moreover, PDT can induce immunogenic cell death (ICD) and improve tumor immunogenicity. However, even though PDT can improve the immunogenicity of TNBC, the inhibitory immune microenvironment of TNBC still weakens the antitumor immune response. Therefore, we used the neutral sphingomyelinase inhibitor GW4869 to inhibit the secretion of small extracellular vesicles (sEVs) by TNBC cells to improve the tumor immune microenvironment and enhance antitumor immunity. In addition, bone mesenchymal stem cell (BMSC)-derived sEVs have good biological safety and a strong drug loading capacity, which can effectively improve the efficiency of drug delivery. In this study, we first obtained primary BMSCs and sEVs, and then the photosensitizers Ce6 and GW4869 were loaded into the sEVs by electroporation to produce immunomodulatory photosensitive nanovesicles (Ce6-GW4869/sEVs). When administered to TNBC cells or orthotopic TNBC models, these photosensitive sEVs could specifically target TNBC and improve the tumor immune microenvironment. Moreover, PDT combined with GW4869-based therapy showed a potent synergistic antitumor effect mediated by direct killing of TNBC and activation of antitumor immunity. Here, we designed photosensitive sEVs that could target TNBC and regulate the tumor immune microenvironment, providing a potential approach for improving the effectiveness of TNBC treatment. Statement of SignificanceWe designed an immunomodulatory photosensitive nanovesicle (Ce6-GW4869/sEVs) with the photosensitizer Ce6 to achieve photodynamic therapy and the neutral sphingomyelinase inhibitor GW4869 to inhibit the secretion of small extracellular vesicles (sEVs) by triple-negative breast cancer (TNBC) cells to improve the tumor immune microenvironment and enhance antitumor immunity. In this study, the immunomodulatory photosensitive nanovesicle could target TNBC cells and regulate the tumor immune microenvironment, thus providing a potential approach for improving the treatment effect in TNBC. We found that the reduction in tumor sEVs secretion induced by GW4869 improved the tumor-suppressive immune microenvironment.Moreover, similar therapeutic strategies can also be applied in other kinds of tumors, especially immunosuppressive tumors, which is of great value for the clinical translation of tumor immunotherapy.

Impact of HepG2 Cells Glutathione Depletion on Neutral Sphingomyelinases mRNA Levels and Activity

Liver cancer is a prevalent form of cancer worldwide. While research has shown that increasing sphingomyelin (SM) hydrolysis by activating the cell surface membrane-associated neutral sphingomyelinase 2 (nSMase2) can control cell proliferation and apoptosis, the role of total glutathione depletion in inducing tumor cell apoptosis via nSMase2 activation is still under investigation. Conversely, glutathione-mediated inhibition of reactive oxygen species (ROS) accumulation is necessary for the enzymatic activity of nSMase1 and nSMase3, increased ceramide levels, and cell apoptosis. This study evaluated the effects of depleting total glutathione in HepG2 cells using buthionine sulfoximine (BSO). The study assessed nSMases RNA levels and activities, intracellular ceramide levels, and cell proliferation using RT-qPCR, Amplex red neutral sphingomyelinase fluorescence assay, and colorimetric assays, respectively. The results indicated a lack of nSMase2 mRNA expression in treated and untreated HepG2 cells. Depletion of total glutathione resulted in a significant increase in mRNA levels but a dramatic reduction in the enzymatic activity of nSMase1 and nSMase3, a rise in ROS levels, a decrease in intracellular levels of ceramide, and an increase in cell proliferation. These findings suggest that total glutathione depletion may exacerbate liver cancer (HCC) and not support using total glutathione-depleting agents in HCC management. It is important to note that these results are limited to HepG2 cells, and further studies are necessary to determine if these effects will also occur in other cell lines. Additional research is necessary to explore the role of total glutathione depletion in inducing tumor cell apoptosis.

Hepatic deletion of serine palmitoyl transferase 2 impairs ceramide/sphingomyelin balance, bile acids homeostasis and leads to liver damage in mice

Ceramides (Cer) have been shown as lipotoxic inducers, which disturb numerous cell-signaling pathways, leading to metabolic disorders such as type 2 diabetes. In this study, we aimed to determine the role of de novo hepatic ceramide synthesis in energy and liver homeostasis in mice. We generated mice lacking serine palmitoyltransferase 2 (Sptlc2), the rate limiting enzyme of ceramide de novo synthesis, in liver under albumin promoter. Liver function, glucose homeostasis, bile acid (BA) metabolism and hepatic sphingolipids content were assessed using metabolic tests and LC-MS. Despite lower expression of hepatic Sptlc2, we observed an increased concentration of hepatic Cer, associated with a 10-fold increase in neutral sphingomyelinase 2 (nSMase2) expression, and a decreased sphingomyelin content in the liver. Sptlc2ΔLiv mice were protected against obesity induced by high fat diet and displayed a defect in lipid absorption. In addition, an important increase in tauro-muricholic acid was associated with a downregulation of the nuclear BA receptor FXR target genes. Sptlc2 deficiency also enhanced glucose tolerance and attenuated hepatic glucose production, while the latter effect was dampened in presence of nSMase2 inhibitor. Finally, Sptlc2 disruption promoted apoptosis, inflammation and progressive development of hepatic fibrosis, worsening with age. Our data suggest a compensatory mechanism to regulate hepatic ceramides content from sphingomyelin hydrolysis, with deleterious impact on liver homeostasis. In addition, our results show the involvement of hepatic sphingolipid modulation in BA metabolism and hepatic glucose production in an insulin-independent manner, which highlight the still under-researched role of ceramides in many metabolic functions.

M6A modification-tuned sphingolipid metabolism regulates postnatal liver development in male mice.

Different organs undergo distinct transcriptional, epigenetic and physiological alterations that guarantee their functional maturation after birth. However, the roles of epitranscriptomic machineries in these processes have remained elusive. Here we demonstrate that expression of RNA methyltransferase enzymes Mettl3 and Mettl14 gradually declines during postnatal liver development in male mice. Liver-specific Mettl3 deficiency causes hepatocyte hypertrophy, liver injury and growth retardation. Transcriptomic and N6-methyl-adenosine (m6A) profiling identify the neutral sphingomyelinase, Smpd3, as a target of Mettl3. Decreased decay of Smpd3 transcripts due to Mettl3 deficiency results in sphingolipid metabolism rewiring, characterized by toxic ceramide accumulation and leading to mitochondrial damage and elevated endoplasmic reticulum stress. Pharmacological Smpd3 inhibition, Smpd3 knockdown or Sgms1 overexpression that counteracts Smpd3 can ameliorate the abnormality of Mettl3-deficent liver. Our findings demonstrate that Mettl3-N6-methyl-adenosine fine-tunes sphingolipid metabolism, highlighting the pivotal role of an epitranscriptomic machinery in coordination of organ growth and the timing of functional maturation during postnatal liver development.

Elevated Estrogen Induces Senescence in Male Endothelial Cells Via Neutral Sphingomyelinase-Mediated Ceramide Formation

Prevalence of cardiovascular disease is higher among trans-females undergoing estrogen therapy compared to cis-females and males. Our previous work has shown that chronic exposure to elevated estrogen induces microvascular endothelial dysfunction in arterioles collected from healthy biological males, however the mechanism contributing to dysfunction is unknown. Conflicting evidence has shown that estrogen can both promote or prevent cell senescence, a phenotypic change characterized by replicative arrest and endothelial dysfunction. Since estrogen is capable of activating neutral sphingomyelinase (NSmase), an enzyme that increases cellular levels of the pro-senescent sphingolipid ceramide, we hypothesized that elevated estrogen induces endothelial cell senescence in biological males through the activation of NSmase. To study this, primary male human umbilical vein endothelial cells (HUVECs) were treated with increasing doses of 17-beta estradiol (E2; 0.5nM to 100nM) for 24hrs with or without a neutral sphingomyelinase inhibitor (GW4869 5μM). Cells were also exposed to C2-ceramide (5μM) or inactive dihidro-C2-ceramide (5μM) and maintained for 48hrs in 1% serum EBM-2 media prior to beta-galactosidase staining (biomarker of senescence). Treatment groups were imaged using 20x bright-field microscopy. Total number of positive beta-galactosidase-stained cells were counted and normalized to total number of cells per image field (mean%±SEM reported; n=3 patients for all); counters were blinded to treatment group. One-Way ANOVA with alpha<0.05 was used to compare between groups. Male HUVECs exhibited no change in percent positive cells with 0.5nM E2 treatment (25±11.4% vs 21.5±2.4% vehicle control), however had significantly higher percentage of positive cells when exposed to higher doses of E2 (1nM, 45.±11.5%; 50nM, 48.1±6.1%; 100nM, 45.5±2.6%; p<0.05 all). Treatment with ceramide (40.44±7.8% vs 11.2±4.3% control, p<0.05) but not dihydroceramide (14.89±12.1%) increased percentage of positive cells. Inhibition of neutral sphingomyelinase alone did not influence percent positivity (12.4±2.7% vs 11.2±4.3% control), however in comparison to 100nM E2 treatment, those treated with GW4869 and 100nM E2 exhibited a lower percentage of positive cells (45.5±2.6% 100nM E2 vs 19.22±7.0% E2+GW4869, p<0.05). These results suggest that elevated estrogen may cause an increase in endothelial cell senescence among biological males through the upregulation of neutral sphingomyelinase-mediated ceramide formation. Our work adds mechanistic insight by which estrogen may promote endothelial dysfunction among trans-females undergoing estrogen therapy and proposes the inhibition of NSmase-mediated ceramide formation as a novel therapeutic avenue for reducing CVD risk associated with gender transition. This work is supported by National Institutes of Health (NHLBI) K08 HL141562 (JKF) and American Heart Association Predoctoral Fellowship 909315 (GSK). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Rab27-dependent extracellular vesicle releasing participates in dengue virus non-structural protein 1 secretion

Abstract Dengue disease is one of important arthropod-borne disease in the world and causes around 20,000 deaths annually. Dengue virus (DENV) non-structural protein 1 (NS1) is the major secreted non-structural protein from infected cells and highly associated with the pathogenesis of severe dengue. Several studies have shown that the secreted NS1 contribute to increase of vascular permeability, coagulopathy, activation of immune cells as well as inflammation. These findings strongly indicate the pathological roles of secreted NS1; however, the secretory pathway is not fully clear. Accumulating evidence have shown that virus-associated extracellular vesicles (EVs) not only contain viral components including viral proteins, genetic material or virion particle but also host components. Rab27 is one of several GTPases essential to regulate EV releasing by affecting docking of multivesicular body (MVB) to the target plasma membrane. Here, we show that NS1 can be exported from DENV-infected cells by EVs. NS1 as well as EV biogenesis-associated proteins ALG-2-interacting protein X (ALIX) and neutral sphingomyelinase2 (nSMase2) co-accumulate in extracellular vesicles. We next determine the topology of NS1 and demonstrate that NS1 is associated at the surface of EVs. In addition, the secretion of DENV NS1 is regulated by Rab27. The EV biogenesis and secretion inhibitor, Tipifarnib treatment decreases the levels of NS1 and nSMase 2 but not the levels of ALIX in the EVs. Taken together, this study not only provide the new insights of mechanisms in DENV NS1 secretion through Rab27-mediated vesicle trafficking but also valuable therapeutic targets to dengue disease. Supported by grants from Ministry of Science and Technology of Taiwan (MOST 111-2320-B-006 -051 -MY3)

Mechanisms of pulmonary microvascular endothelial cells barrier dysfunction induced by LPS: The roles of ceramides and the Txnip/NLRP3 inflammasome.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) are characterized by pulmonary microvascular endothelial cells (PMVECs) barrier dysfunction and proinflammatory cytokine influx into lung tissue, resulting in pulmonary oedema. Ceramide overproduction is an important mediator of pulmonary hyperinflammation and pulmonary oedema in Acute lung injury (ALI). Ceramides induce NLRP3 inflammasome activation are essential for the hyperinflammatory response. However, the roles and specific mechanisms of ceramide-induced NLRP3 inflammasome activation, proinflammatory cytokine manufacturing and PMVECs barrier dysfunction in ALI are unclear. Herein, pretreatment with the acid sphingomyelinase (ASMase) inhibitor imipramine (but not a neutral sphingomyelinase (NSMase) inhibitor or de novo pathway inhibitor) significantly inhibited ceramide early production in rats with lipopolysaccharide (LPS)-induced ALI; Furthermore, the Txnip/NLRP3 inflammasome activation, proinflammatory cytokine release, increased PMVECs permeability and lung injury were significantly decreased. Verapamil, a Txnip inhibitor, substantially inhibited Txnip/NLRP3 inflammasome activation, proinflammatory cytokine release, increased PMVECs permeability and lung injury in rats with C8-ceramide-induced ALI. In vitro, short hairpin RNA-mediated Txnip silencing significantly inhibited C8-ceramide-induced Txnip/NLRP3 inflammasome activation in NR8383 alveolar macrophages (AMs) and early secretion of the proinflammatory cytokines IL-1β (4-12h) as well as IL-6 and TNF-α at subsequent times (later than 12h). However, C8-ceramide significantly increased the early secretion (within 8h) of the proinflammatory cytokines IL-1β, IL-6 and TNF-α in a co-culture model of NR8383 AMs and PMVECs, and Txnip silencing of NR8383 AMs inhibited the secretion of pro-inflammatory cytokines and reduced cytoskeletal rearrangements, intercellular connection breakage and hyperpermeability in PMVECs. Overall, our results suggest that in LPS-induced ALI, ceramide-mediated Txnip/NLRP3 inflammasome activation in NR8383 AMs leads to early IL-1β release, subsequently inducing PMVECs injury and release of the proinflammatory cytokines IL-6 and TNF-α, ultimately leading to PMVECs barrier dysfunction and ALI.

Quercetin Affects Ceramide but not Sphingosine-1-phosphate in ras-transformed 3T3 Fibroblasts

Investigations were carried out on the effect of quercetin on the level of the major sphingolipid metabolites in ras-transformed 3T3 fibroblasts. Quercetin is a polyphenol of a significant biomedical importance, which has been reported to show beneficial effects such as antioxidant, anti-neoplastic, anti-ageing, etc. Sphingolipids are functionally active lipid molecules, which regulate important cellular processes, like proliferation, apoptosis and transmembrane signalling, among others. Despite the numerous investigations devoted to the influence of quercetin on the lipid metabolism of various types of cancer cells, the mechanism of this influence on the major sphingolipid pathways still remains unclear. Our studies showed that sphingomyelin was decreased in ras-transformed 3T3 fibroblasts, whereas ceramide, which is a pro-apoptotic factor, was increased as a result of elevated neutral sphingomyelinase activity. However, the level of sphingosine-1-phosphate, which is related to cell proliferation, was not altered as a result of quercetin treatment, which is an unexpected finding when compared to our previous studies with cancer cells. In addition, the observed changes in the balancing enzyme in the sphingolipid pathway, sphingosine kinase 1, which produces sphingosine-1-phosphate, were not statistically significant in quercetin-treated oncogene-transformed fibroblasts. The presented data are useful for better understanding of the effect of flavonoids on the regulation of sphingolipid metabolism and could help in the development of complex antitumour therapeutic approaches, involving natural antitumour agents like polyphenols.

Acetylcysteine modulates sphingolipid levels by inhibiting neutral sphingomyelinase during aging in rat tissues

Old-age-associated pathologies usually coincide with altered sphingolipid metabolism. Changes in the level of sphingomyelin (SM) and ceramide are largely determined by the activation of neutral sphingomyelinase (nSMase) during aging. nSMase is a key enzyme in sphingolipid turnover; it generates ceramide through the hydrolysis of SM. While N—acetylcysteine (NAC) is a powerful antioxidant which has anti-inflammatory properties and it is the precursor of glutathione (GSH). The aim of this study is to determine the impact of NAC on nSMase activity in the age-dependent changes of sphingolipid (SM and ceramide) levels and GSH content in blood serum, gastrocnemius muscle and kidney tissue of 24-month-old rats. It was found that the effect of NAC on the tissue of 24-month-old rats increased SM content; GSH content and decreased the ceramide content. The gastrocnemius muscle and kidney were the most sensitive to the effects of NAC. The results show that GSH inhibit the activity of nSMase. These results emphasize that nSMase. plays a key role in age-associated ceramide accumulation in rat tissues during aging. Furthermore, it was indicated that NAC is an effective modulator of nSMase and has pleiotropic effects that may alter sphingolipid metabolism. The nSMase inhibition enhances the benefits of NAC treatment.

Personalized precision medicine of strategic health action in Niemann-Pick type A/B disease: Case report

Niemann-Pick Diseases (NPD) is lysosomal storage diseases caused by Acid Sphingo Myelinase (ASM) deficiency, which catalyzes the hydrolysis of Sphingo Myelin

The Effect of Cholesterol in MCF7 Human Breast Cancer Cells

In the last decade, cholesterol level has been implicated in several types of cancer, including breast cancer. In the current study, we aimed to investigate the condition of lipid depletion, hypocholesterolemia or hypercholesterolemia reproduced in vitro to analyze the response of different human breast cancer cells. Thus, MCF7 as the luminal A model, MB453 as the HER2 model and MB231 as the triple-negative model were used. No effect on cell growth and viability was detected in MB453 and MB231 cells. In MCF7 cells, hypocholesterolemia (1) reduced cell growth, and Ki67 expression; (2) increased ER/PgR expression; (3) stimulated the 3-Hydroxy-3-Methylglutaryl-CoA reductase and neutral sphingomyelinase and; (4) stimulated the expression of CDKN1A gene coding cyclin-dependent kinase inhibitor 1A protein, GADD45A coding growth arrest and DNA-damage-inducible alpha protein and, PTEN gene coding phosphatase and tensin homolog. All these effects were exacerbated by the lipid-depleted condition and reversed by the hypercholesterolemic condition. The relationship between cholesterol level and sphingomyelin metabolism was demonstrated. In summary, our data suggest that cholesterol levels should be controlled in luminal A breast cancer.

Electrical Stimulation Increases the Secretion of Cardioprotective Extracellular Vesicles from Cardiac Mesenchymal Stem Cells.

Clinical trials have shown that electric stimulation (ELSM) using either cardiac resynchronization therapy (CRT) or cardiac contractility modulation (CCM) approaches is an effective treatment for patients with moderate to severe heart failure, but the mechanisms are incompletely understood. Extracellular vesicles (EV) produced by cardiac mesenchymal stem cells (C-MSC) have been reported to be cardioprotective through cell-to-cell communication. In this study, we investigated the effects of ELSM stimulation on EV secretion from C-MSCs (C-MSCELSM). We observed enhanced EV-dependent cardioprotection conferred by conditioned medium (CM) from C-MSCELSM compared to that from non-stimulated control C-MSC (C-MSCCtrl). To investigate the mechanisms of ELSM-stimulated EV secretion, we examined the protein levels of neutral sphingomyelinase 2 (nSMase2), a key enzyme of the endosomal sorting complex required for EV biosynthesis. We detected a time-dependent increase in nSMase2 protein levels in C-MSCELSM compared to C-MSCCtrl. Knockdown of nSMase2 in C-MSC by siRNA significantly reduced EV secretion in C-MSCELSM and attenuated the cardioprotective effect of CM from C-MSCELSM in HL-1 cells. Taken together, our results suggest that ELSM-mediated increases in EV secretion from C-MSC enhance the cardioprotective effects of C-MSC through an EV-dependent mechanism involving nSMase2.

Virtual screening reveals aprepitant to be a potent inhibitor of neutral sphingomyelinase 2: implications in blockade of exosome release in cancer therapy.

Exosomes are membrane-derived nano-vesicles upregulated in pathological conditions like cancer. Therefore, inhibiting their release is a potential strategy for the development of more efficient combination therapies. Neutral sphingomyelinase 2 (nSMase2) is a key component in exosome release; however, a clinically safe yet efficient nSMase2 inhibitor remains to be used discovered. Accordingly, we made an effort to identify potential nSMase2 inhibitor(s) among the approved drugs. Virtual screening was performed and aprepitant was selected for further investigation. To evaluate the reliability of the complex, molecular dynamics were performed. Finally, using the CCK-8 assay in HCT116 cells, the highest non-toxic concentrations of aprepitant were identified and the nSMase2 activity assay was performed to measure the inhibitory activity of aprepitant, in vitro. To validate the screening results, molecular docking was performed, and the retrieved scores were in line with the screening results. The root-mean-square deviation (RMSD) plot of aprepitant-nSMase2 showed proper convergence. Following treatment with different concentrations of aprepitant in both cell-free and cell-dependent assays, nSMase2 activity was remarkably decreased. Aprepitant, at a concentration as low as 15µM, was able to inhibit nSmase2 activity in HCT116 cells without any significant effects on their viability. Aprepitant is therefore suggested to be a potentially safe exosome release inhibitor.

Ceramide-dependent trafficking of Epstein-Barr virus LMP1 to small extracellular vesicles

Epstein-Barr virus (EBV) is a human herpesvirus that is associated with a multitude of cancers. The primary EBV oncogene latent membrane protein 1 (LMP1) is secreted from infected cancer cells in small extracellular vesicles (EVs). Additionally, the tetraspanin protein CD63 forms a complex with LMP1 and CD63 can be trafficked to EVs through a ceramide-dependent manner. Therefore, we hypothesize that ceramide is required for efficient packaging of LMP1 into small EVs. Following treatment with the neutral sphingomyelinase inhibitor GW4869, LMP1 cellular localization was disrupted and immunoblotting of EV lysates revealed a significant reduction in extracellular LMP1. NTA of EVs from the LCLs treated with GW4869 demonstrated a significant decrease in particle secretion. Additionally, ceramide inhibition resulted in enhanced LMP1-mediated NFkB activation in EV producing cells. Taken together, these data reveal a critical role for the lipid ceramide in LMP1 exosomal trafficking and the oncogenic signaling properties of the viral protein.

Mechanisms of Arachidonic Acid In Vitro Tumoricidal Impact.

To promote the potential of arachidonic acid (ARA) for cancer prevention and management, experiments were implemented to disclose the mechanisms of its tumoricidal action. Hepatocellular, lung, and breast carcinoma and normal hepatocytes cell lines were exposed to 0 or 50 μM ARA for 30 min and then assessed for proliferative capacity, surface membrane-associated sphingomyelin (SM) content, neutral sphingomyelinase (nSMase) activity, beta 2 microglobulin (β2 m) expression, and ceramide (Cer) levels. Reactive oxygen species (ROS) content and caspase 3/7 activity were evaluated. Exposure to ARA for 30 min led to impairment of the tumor cells' proliferative capacity and revealed that the different cell lines display remarkably similar surface membrane SM content but diverse responses to ARA treatment. Arachidonic acid tumoricidal impact was shown to be associated with nSMase activation, exposure of cell surface membrane β2 m to antibody binding, and hydrolysis of SM to Cer, which accumulated on the cell surface and in the cytosol. The ARA and Cer-mediated inhibition of tumor cell viability appeared to be independent of ROS generation or caspase 3/7 activation. The data were compared and contrasted to findings reported in the literature on ARA tumoricidal mechanisms.

SMPD4 regulates mitotic nuclear envelope dynamics and its loss causes microcephaly and diabetes.

Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder with progressive congenital microcephaly and early death. SMPD4 encodes a sphingomyelinase that hydrolyses sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes (NPC). We refine the clinical phenotype of loss-of-function SMPD4 variants by describing five individuals from three unrelated families with longitudinal data due to prolonged survival. All individuals surviving beyond infancy developed insulin-dependent diabetes, besides presenting with a severe neurodevelopmental disorder and microcephaly, making diabetes one of the most frequent age-dependent non-cerebral abnormalities. We studied the function of SMPD4 at the cellular and organ levels. Knock-down of SMPD4 in human neural stem cells causes reduced proliferation rates and prolonged mitosis. Moreover, SMPD4 depletion results in abnormal nuclear envelope breakdown and reassembly during mitosis and decreased post-mitotic NPC insertion. Fibroblasts from affected individuals show deficient SMPD4-specific neutral sphingomyelinase activity, without changing (sub)cellular lipidome fractions, which suggests a local function of SMPD4 on the nuclear envelope. In embryonic mouse brain, knockdown of Smpd4 impairs cortical progenitor proliferation and induces premature differentiation by altering the balance between neurogenic and proliferative progenitor cell divisions. We hypothesize that, in individuals with SMPD4-related disease, nuclear envelope bending, which is needed to insert NPCs in the nuclear envelope, is impaired in the absence of SMPD4 and interferes with cerebral corticogenesis and survival of pancreatic beta cells.