Sphingomyelinase Activity Research Articles

Acid sphingomyelinase deficiency protects mitochondria and improves function recovery after brain injury

Traumatic brain injury (TBI) is one of the leading causes of disability worldwide and a prominent risk factor for neurodegenerative diseases. The expansion of nervous tissue damage after the initial trauma involves a multifactorial cascade of events, including excitotoxicity, oxidative stress, inflammation, and deregulation of sphingolipid metabolism that further mitochondrial dysfunction and secondary brain damage. Here, we show that a posttranscriptional activation of an acid sphingomyelinase (ASM), a key enzyme of the sphingolipid recycling pathway, resulted in a selective increase of sphingosine in mitochondria during the first week post-TBI that was accompanied by reduced activity of mitochondrial cytochrome oxidase and activation of the Nod-like receptor protein 3 inflammasome. TBI-induced mitochondrial abnormalities were rescued in the brains of ASM KO mice, which demonstrated improved behavioral deficit recovery compared with WT mice. Furthermore, an elevated autophagy in an ASM-deficient brain at the baseline and during the development of secondary brain injury seems to foster the preservation of mitochondria and brain function after TBI. Of note, ASM deficiency attenuated the early stages of reactive astrogliosis progression in an injured brain. These findings highlight the crucial role of ASM in governing mitochondrial dysfunction and brain-function impairment, emphasizing the importance of sphingolipids in the neuroinflammatory response to TBI.

Small-hairpin RNA and pharmacological targeting of neutral sphingomyelinase prevent diaphragm weakness in rats with heart failure and reduced ejection fraction.

Heart failure with reduced ejection fraction (HFREF) increases neutral sphingomyelinase (NSMase) activity and mitochondrial reactive oxygen species (ROS) emission and causes diaphragm weakness. We tested whether a systemic pharmacological NSMase inhibitor or short-hairpin RNA (shRNA) targeting NSMase isoform 3 (NSMase3) would prevent diaphragm abnormalities induced by HFREF caused by myocardial infarction. In the pharmacological intervention, we used intraperitoneal injection of GW4869 or vehicle. In the genetic intervention, we injected adeno-associated virus serotype 9 (AAV9) containing shRNA targeting NSMase3 or a scrambled sequence directly into the diaphragm. We also studied acid sphingomyelinase-knockout mice. GW4869 prevented the increase in diaphragm ceramide content, weakness, and tachypnea caused by HFREF. For example, maximal specific forces (in N/cm2) were vehicle [sham 31 ± 2 and HFREF 26 ± 2 ( P < 0.05)] and GW4869 (sham 31 ± 2 and HFREF 31 ± 1). Respiratory rates were (in breaths/min) vehicle [sham 61 ± 3 and HFREF 84 ± 11 ( P < 0.05)] and GW4869 (sham 66 ± 2 and HFREF 72 ± 2). AAV9-NSMase3 shRNA prevented heightening of diaphragm mitochondrial ROS and weakness [in N/cm2, AAV9-scrambled shRNA: sham 31 ± 2 and HFREF 27 ± 2 ( P < 0.05); AAV9-NSMase3 shRNA: sham 30 ± 1 and HFREF 30 ± 1] but displayed tachypnea. Both wild-type and ASMase-knockout mice with HFREF displayed diaphragm weakness. Our study suggests that activation of NSMase3 causes diaphragm weakness in HFREF, presumably through accumulation of ceramide and elevation in mitochondrial ROS. Our data also reveal a novel inhibitory effect of GW4869 on tachypnea in HFREF likely mediated by changes in neural control of breathing.

Protective role of epigallocatechin-3-gallate in NADPH oxidase-MMP2-Spm-Cer-S1P signalling axis mediated ET-1 induced pulmonary artery smooth muscle cell proliferation.

The signalling pathway involving MMP-2 and sphingosine-1-phosphate (S1P) in endothelin-1 (ET-1) induced pulmonary artery smooth muscle cell (PASMC) proliferation is not clearly known. We, therefore, investigated the role of NADPH oxidase derived O2.--mediated modulation of MMP2-sphingomyeline-ceramide-S1P signalling axis in ET-1 induced increase in proliferation of PASMCs. Additionally, protective role of the tea cathechin, epigallocatechin-3-gallate (EGCG), if any, in this scenario has also been explored. ET-1 markedly increased NADPH oxidase and MMP-2 activities and proliferation of bovine pulmonary artery smooth muscle cells (BPASMCs). ET-1 also caused significant increase in sphingomyelinase (SMase) activity, ERK1/2 and sphingosine kinase (SPHK) phosphorylations, and S1P level in the cells. EGCG inhibited ET-1 induced increase in SMase activity, ERK1/2 and SPHK phosphorylations, S1P level and the SMC proliferation. EGCG also attenuated ET-1 induced activation of MMP-2 by inhibiting NADPH oxidase activity upon inhibiting the association of the NADPH oxidase components, p47phox and p67phox in the cell membrane. Molecular docking study revealed a marked binding affinity of p47phox with the galloyl group of EGCG. Overall, our study suggest that ET-1 induced proliferation of the PASMCs occurs via NADPH oxidase-MMP2- Spm- Cer-S1P signalling axis, and EGCG attenuates ET-1 induced increase in proliferation of the cells by inhibiting NADPH oxidase activity.

Exosomes mediate Zika virus transmission through SMPD3 neutral Sphingomyelinase in cortical neurons

ABSTRACT The harmful effects of ZIKA virus (ZIKV) infection are reflected by severe neurological manifestations such as microcephaly in neonates and other complications associated with Guillain-Barré syndrome in adults. The transmission dynamics of ZIKV in or between neurons, or within the developing brains of the foetuses are not fully understood. Using primary cultures of murine cortical neurons, we show that ZIKV uses exosomes as mediators of viral transmission between neurons. Cryo-electron microscopy showed heterogeneous population of neuronal exosomes with a size range of 30–200 nm. Increased production of exosomes from neuronal cells was noted upon ZIKV infection. Neuronal exosomes contained both ZIKV viral RNA and protein(s) that were highly infectious to naïve cells. RNaseA and neutralizing antibodies treatment studies suggest the presence of viral RNA/proteins inside exosomes. Exosomes derived from time- and dose-dependent incubations showed increasing viral loads suggesting higher packaging and delivery of ZIKV RNA and proteins. Furthermore, we noted that ZIKV induced both activity and gene expression of neutral Sphingomyelinase (nSMase)-2/SMPD3, an important molecule that regulates production and release of exosomes. Silencing of SMPD3 in neurons resulted in reduced viral burden and transmission through exosomes. Treatment with SMPD3 specific inhibitor GW4869, significantly reduced ZIKV loads in both cortical neurons and in exosomes derived from these neuronal cells. Taken together, our results suggest that ZIKV modulates SMPD3 activity in cortical neurons for its infection and transmission through exosomes perhaps leading to severe neuronal death that may result in neurological manifestations such as microcephaly in the developing embryonic brains.

Hemolysins of &amp;lt;i&amp;gt;Staphylococcus aureus&amp;lt;/i&amp;gt;—An Update on Their Biology, Role in Pathogenesis and as Targets for Anti-Virulence Therapy

Staphylococcus aureus is a dangerous gram positive bacterial pathogen which, not only evades the host’s immune system but also can destroy the leucocytes especially neutrophils. It has an embodiment of virulence factors most of which are secreted. Staphylococcus aureus secretes a number of toxins which cause tissue damage and facilitate spreading and nutrients uptake. Among the toxins, hemolysins α, β, γ, δ and Panton Valentine Leukocidin (PVL) are unique that they drill pores in the membrane, leading to the efflux of vital molecules and metabolites. Hemolysins also help in the scavenging of iron, although many of them also have leucolytic properties. α-hemolysin, also known as α-toxin, is the most prominent cytotoxin which damages a wide range of host cells including epithelial cells, endothelial cells, erythrocytes, monocytes, keratinocytes and it damages cell membrane and induces apoptosis. β-Hemolysin significantly affects human immune cell function. It has Mg2+ dependent sphingomyelinase activity and degrades sphingomyelin of plasma membrane into phosphorylcholine and ceramides. The bi-component leukocidins, which include γ-hemolysin and PVL, attack human phagocytic cells and greatly contribute to immune evasion. Delta toxin is a low molecular weight exotoxin with a broad cytolytic activity. Virulence determinants, quorum sensing and biofilm synthesis provide some attractive targets for design and development of a new group of antimicrobial compounds. This review provides an update on the structure, biological functions of hemolysins and their role in quorum sensing/biofilm synthesis (if any) and as effective therapeutic targets for anti-virulence drug development. We have tried to bring together information available on various aspects of hemolysins and highlighted their distribution among all species of Staphylococcus and other bacteria. We have updated the status of development of candidate drugs targeting the hemolysins for anti-virulence therapy as it offers an additional strategy to reduce the severity of infection and which would, through quorum quenching, delay the development biofilms leading to drug resistance.

Human Remyelination Promoting Antibody Stimulates Astrocytes Proliferation Through Modulation of the Sphingolipid Rheostat in Primary Rat Mixed Glial Cultures.

Remyelination promoting human IgMs effectively increase the number of myelinated axons in animal models of multiple sclerosis. Hence, they ultimately stimulate myelin production by oligodendrocytes (OLs); however, their exact mechanism of action remains to be elucidated, and in particular, it remains unclear whether they are directly targeting OLs, or their action is mediated by effects on other cell types. We assessed the effect of remyelination promoting antibody rHIgM22 on the proliferative response and on the ceramide/sphingosine 1-phosphate rheostat in mixed glial cell cultures (MGCs). rHIgM22 treatment caused a time-dependent increase in PDGFαR protein in MGCs. Forty-eight hours of treatment with rHIgM22 induced a dose-dependent proliferative response (evaluated as total cell number and as EdU(+) cell number) in MGCs. When the proliferation response of MGCs to rHIgM22 was analyzed as a function of the cell types, the most significant proliferative response was associated with GLAST(+) cells, i.e., astrocytes. In many cell types, the balance between different sphingolipid mediators (the "sphingolipid rheostat"), in particular ceramide and sphingosine 1-phosphate, is critical in determining the cell fate. rHIgM22 treatment in MGCs induced a moderate but significant inhibition of total acidic sphingomyelinase activity (measured in vitro on cell lysates), the main enzyme responsible for the stimulus-mediated production of ceramide, when treatment was performed in serum containing medium, but no significant differences were observed when antibody treatment was performed in the absence of serum. Moreover, rHIgM22 treatment, either in the presence or in absence of serum, had no effects on ceramide levels. On the other hand, rHIgM22 treatment for 24h induced increased production and release of sphingosine 1-phosphate in the extracellular milieu of MGC. Release of sphingosine 1-phosphate upon rHIgM22 treatment was strongly reduced by a selective inhibitor of PDGFαR. Increased sphingosine 1-phosphate production does not seem to be mediated by regulation of the biosynthetic enzymes, sphingosine kinase 1 and 2, since protein levels of these enzymes and phosphorylation of sphingosine kinase 1 were unchanged upon rHIgM22 treatment. Instead, we observed a significant reduction in the levels of sphingosine 1-phosphate lyase 1, one of the key catabolic enzymes. Remarkably, rHIgM22 treatment under the same experimental conditions did not induce changes in the production and/or release of sphingosine 1-phosphate in pure astrocyte cultures. Taken together, these data suggest that rHIgM22 indirectly influences the proliferation of astrocytes in MGCs, by affecting the ceramide/sphingosine 1-phosphate balance. The specific cell population directly targeted by rHIgM22 remains to be identified, however our study unveils another aspect of the complexity of rHIgM22-induced remyelinating effect.

Peripheral Acid Sphingomyelinase Activity Is Associated with Biomarkers and Phenotypes of Alcohol Use and Dependence in Patients and Healthy Controls.

By catalyzing the hydrolysis of sphingomyelin into ceramide, acid sphingomyelinase (ASM) changes the local composition of the plasma membrane with effects on receptor-mediated signaling. Altered enzyme activities have been noted in common human diseases, including alcohol dependence. However, the underlying mechanisms remain largely unresolved. Blood samples were collected from early-abstinent alcohol-dependent in-patients (n[♂] = 113, n[♀] = 87) and matched healthy controls (n[♂] = 133, n[♀] = 107), and analyzed for routine blood parameters and serum ASM activity. We confirmed increased secretory ASM activities in alcohol-dependent patients compared to healthy control subjects, which decreased slightly during detoxification. ASM activity correlated positively with blood alcohol concentration, withdrawal severity, biomarkers of alcohol dependence (liver enzyme activities of gamma-glutamyl transferase, alanine aminotransferase, aspartate aminotransferase; homocysteine, carbohydrate-deficient transferrin; mean corpuscular volume, and creatine kinase). ASM activity correlated negatively with leukocyte and thrombocyte counts. ASM and gamma-glutamyl transferase were also associated in healthy subjects. Most effects were similar for males and females with different strengths. We describe previously unreported associations between ASM activity and markers of liver damage and myelosuppression. Further research should investigate whether this relationship is causal, or whether these parameters are part of a common pathway in order to gain insights into underlying mechanisms and develop clinical applications.

On the Importance of the C(1)-OH and C(3)-OH Functional Groups of the Long-Chain Base of Ceramide for Interlipid Interaction and Lateral Segregation into Ceramide-Rich Domains.

Ceramides are important intermediates in sphingolipid biosynthesis (and degradation) and are normally present in only small amounts in unstressed cells. However, following the receptor-mediated activation of neutral sphingomyelinase, sphingomyelin can acutely give rise to substantial amounts of ceramides, which dramatically alter membrane properties. In this study, we have examined the role of the 1-OH and 3-OH functional groups of ceramide for its membrane properties. We have specifically examined how the oxidation of the primary alcohol to COOH or COOMe in palmitoyl ceramide (PCer) or the removal of either the primary alcohol or C(3)-OH (deoxy analogs) affected ceramides' interlipid interactions in fluid phosphatidylcholine bilayers. Measuring the time-resolved fluorescence emission of trans-parinaric acid, or its steady-state anisotropy, we have obtained information about the propensity of the ceramide analogs to form ceramide-rich domains and the thermostability of the formed domains. We observed that the oxidation of the primary alcohol to COOH shifted the ceramide's gel-phase onset concentration to slightly higher values in 1-palmitoyl-2-oleoyl- sn-3- glycero-3-phosphocholine (POPC) bilayers. Methylation of the COOH function of the ceramide did not change the segregation tendency further. The complete removal of the primary alcohol dramatically reduced the ability of 1-deoxy-PCer to form ceramide-rich ordered domains. However, the removal 3-OH (in 3-deoxy-PCer) had only small effects on the lateral segregation of the ceramide analog. The thermostability of the ceramide-rich domains in the POPC bilayers decreased in the following order: 1-OH > COOH > COOMe = 3-deoxy > 1-deoxy. We conclude that ceramide needs a hydrogen-bonding-competent functional group in the C(1) position to be able to form laterally segregated ceramide-rich domains of high packing density in POPC bilayers. The presence or absence of 3-OH was not functionally critical for ceramide's lateral segregation properties.

Acid sphingomyelinase: No potential as a biomarker for multiple sclerosis

BackgroundMultiple sclerosis (MS) lacks reliable biomarkers that reflect disease activity. Recent evidence suggests that an altered sphingolipid metabolism is associated with MS pathogenesis. ObjectiveTo explore acid sphingomyelinase (ASM) activity and altered sphingolipid metabolism as potential biomarkers in serum of MS patients, to predict active and progressive disease, and response to disease modifying therapy (DMT). MethodsLevels of serum ASM activity were longitudinally analyzed in 40 clinically isolated syndrome, 64 relapsing remitting (RR) and 10 primary progressive MS patients, and 22 healthy controls (HC). ASM activity and sphingolipid levels were measured in a different sample of 61 RRMS patients using DMT. ResultsA significant difference in ASM activity levels was observed between MS patients and HC (p < 0.001). There was no correlation between ASM activity levels and disease activity, progression or response to DMT. Ceramide (Cer)-C16:0 , Cer-C24:0 and sphingomyelin (SM)-C20:0, SM-C22:0, SM-C24:0 and SM-C24:1 showed a significant increase during fingolimod use. ConclusionAlthough higher levels in MS patients were found, ASM activity levels do not show potential as a biomarker for predicting disease activity, progression or response to DMT. Two ceramides and four types of sphingomyelin require further investigation as potential markers for treatment response.

Early postoperative changes of sphingomyelins and ceramides after laparoscopic sleeve gastrectomy

BackgroundThis study aimed to determine early postoperative changes of serum sphingomyelin (SM) and ceramide (CER) species following laparoscopic sleeve gastrectomy (LSG).MethodsTwenty obese patients [mean body mass index (BMI) 45,64 ± 6,10 kg/m2] underwent LSG and normal weight control patients (mean BMI 31,51 ± 6,21 kg/m2) underwent laparoscopic cholecystectomy. Fasting blood samples were collected prior to surgery, at day 1 and day 30 after surgery. Circulating levels of C16-C24 SMs, C16-C24 CERs and sphingosine-1-phosphate (S1P) were determined by an optimized multiple reaction monitoring (MRM) method using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Serum activity of neutral sphingomyelinase (N-SMase) was assayed by standard kit methods, and ceramide-1-phosphate (C1P) levels were determined by enzyme-linked immunosorbent assay (ELISA). Lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods. Insulin sensitivity was evaluated using homeostatic model assessment for insulin resistance (HOMA IR).ResultsA significant decrease was observed in serum levels of very-long-chain C24 SM, very-long-chain C22-C24 CERs, HOMA-IR, N-SMase and C1P in LSG patients after postoperation day 1 and day 30 compared to preoperation levels. At 30 days postsurgery, BMI was reduced by 11%, fasting triglycerides were significantly decreased, and insulin sensitivity was increased compared to presurgery values. A significant positive correlation was found between HOMA-IR and serum levels of C22-C24 CERs in LSG patients.ConclusionWe conclude that very long chain CERs may mediate improved insulin sensitivity after LSG.

CD40 Enhances Sphingolipids in Orbital Fibroblasts: Potential Role of Sphingosine-1-Phosphate in Inflammatory T-Cell Migration in Graves' Orbitopathy.

Graves' orbitopathy (GO) is an autoimmune orbital disorder associated with Graves' disease caused by thyrotropin receptor autoantibodies. Orbital fibroblasts (OFs) and CD40 play a key role in disease pathogenesis. The bioactive lipid sphingosine-1-phosphate (S1P) has been implicated in promoting adipogenesis, fibrosis, and inflammation in OFs. We investigated the role of CD40 signaling in inducing S1P activity in orbital inflammation. OFs and T cells were derived from GO patients and healthy control (Ctl) persons. S1P abundance in orbital tissues was evaluated by immunofluorescence. OFs were stimulated with CD40 ligand and S1P levels were determined by ELISA. Further, activities of acid sphingomyelinase (ASM), acid ceramidase, and sphingosine kinase were measured by ultraperformance liquid chromatography. Sphingosine and ceramide contents were analyzed by mass spectrometry. Finally, the role for S1P in T-cell attraction was investigated by T-cell migration assays. GO orbital tissue showed elevated amounts of S1P as compared to control samples. Stimulation of CD40 induced S1P expression in GO-derived OFs, while Ctl-OFs remained unaffected. A significant increase of ASM and sphingosine kinase activities, as well as lipid formation, was observed in GO-derived OFs. Migration assay of T cells in the presence of SphK inhibitor revealed that S1P released by GO-OFs attracted T cells for migration. The results demonstrated that CD40 ligand stimulates GO fibroblast to produce S1P, which is a driving force for T-cell migration. The results support the use of S1P receptor signaling modulators in GO management.

Nuclear Lipid Microdomains Regulate Daunorubicin Resistance in Hepatoma Cells.

Daunorubicin is an anticancer drug, and cholesterol is involved in cancer progression, but their relationship has not been defined. In this study, we developed a novel experimental model that utilizes daunorubicin, cholesterol, and daunorubicin plus cholesterol in the same cells (H35) to search for the role of nuclear lipid microdomains, rich in cholesterol and sphingomyelin, in drug resistance. We find that the daunorubicin induces perturbation of nuclear lipid microdomains, localized in the inner nuclear membrane, where active chromatin is anchored. As changes of sphingomyelin species in nuclear lipid microdomains depend on neutral sphingomyelinase activity, we extended our studies to investigate whether the enzyme is modulated by daunorubicin. Indeed the drug stimulated the sphingomyelinase activity that induced reduction of saturated long chain fatty acid sphingomyelin species in nuclear lipid microdomains. Incubation of untreated-drug cells with high levels of cholesterol resulted in the inhibition of sphingomyelinase activity with increased saturated fatty acid sphingomyelin species. In daunodubicin-treated cells, incubation with cholesterol reversed the action of the drug by acting via neutral sphingomyelinase. In conclusion, we suggest that cholesterol and sphingomyelin-forming nuclear lipid microdomains are involved in the drug resistance.

Chronic Psychosocial Stress in Mice Is Associated With Increased Acid Sphingomyelinase Activity in Liver and Serum and With Hepatic C16:0-Ceramide Accumulation.

Chronic psychosocial stress adversely affects human morbidity and is a risk factor for inflammatory disorders, liver diseases, obesity, metabolic syndrome, and major depressive disorder (MDD). In recent studies, we found an association of MDD with an increase of acid sphingomyelinase (ASM) activity. Thus, we asked whether chronic psychosocial stress as a detrimental factor contributing to the emergence of MDD would also affect ASM activity and sphingolipid (SL) metabolism. To induce chronic psychosocial stress in male mice we employed the chronic subordinate colony housing (CSC) paradigm and compared them to non-stressed single housed control (SHC) mice. We determined Asm activity in liver and serum, hepatic SL concentrations as well as hepatic mRNA expression of genes involved in SL metabolism. We found that hepatic Asm activity was increased by 28% (P = 0.006) and secretory Asm activity by 47% (P = 0.002) in stressed mice. C16:0-Cer was increased by 40% (P = 0.008). Gene expression analysis further revealed an increased expression of tumor necrosis factor (TNF)-α (P = 0.009) and of several genes involved in SL metabolism (Cers5, P = 0.028; Cers6, P = 0.045; Gba, P = 0.049; Gba2, P = 0.030; Ormdl2, P = 0.034; Smpdl3B; P = 0.013). Our data thus provides first evidence that chronic psychosocial stress, at least in mice, induces alterations in SL metabolism, which in turn might be involved in mediating the adverse health effects of chronic psychosocial stress and peripheral changes occurring in mood disorders.

Deep sequencing of SMPD1 gene revealed a heterozygous frameshift mutation (p.Ser192Alafs) in a Palestinian infant with Niemann\u2013Pick disease type A: a case report

BackgroundNiemann–Pick disease is caused by reduced level of the lysosomal enzyme acid sphingomyelinase. Children can survive between 2 and 12 years based on the disease type. Two main types are well known: type A and B. Niemann–Pick disease type A is characterized by severe central nervous system deterioration and hepatosplenomegaly while type B is a progressive hypersplenism accompanied with gradual deterioration of pulmonary function.Case presentationWe describe an 11-month-old Palestinian baby boy with hepatosplenomegaly, hypotonia, delayed motor development, laryngomalacia, bilateral cherry-red spots, and failure to thrive. Metabolic screening, blood count, differential tests, immunology screen, infectious disease screen, urine, biochemical tests as well as molecular diagnosis were performed. The molecular diagnosis was done by amplifying the whole sphingomyelin phosphodiesterase 1 (SMPD1) gene, followed by deep sequencing. The obtained sequences were aligned, de novo assembled and compared to human reference gene (GenBank GeneID: NG_011780.1, Ensembl version ENSG00000166311 and protein identified as UniProtKB – P17405).Two known mutations were identified in our patient: the pathogenic frameshift mutation NM_000543.4(SMPD1):c.573delT (p.Ser192Alafs) and the benign polymorphism NM_000543.4(SMPD1):c.107T>C (p.Val36Ala). The enzyme study showed a very low level of enzymatic activity of acidic sphingomyelinase (0.1 nmol/ml per hour). Correlations between clinical findings, laboratory data, and sequence analysis are presented.ConclusionsIn conclusion, this is the first report about a heterozygote frameshift p.Ser192AlafsX65 in a Palestinian patient with Niemann–Pick disease type A, emphasizing the importance of deep sequencing in genetic diagnosis of this rare inherited disease.

Derivatization of common antidepressant drugs increases inhibition of acid sphingomyelinase and reduces induction of phospholipidosis.

In recent studies, major depressive disorder (MDD) was linked to an increase in acid sphingomyelinase (ASM) activity. Several drugs that are commonly used to treat MDD functionally inhibit the lysosomal enzyme ASM and are called functional inhibitors of ASM (FIASMAs). These drugs are classified as cationic amphiphilic drugs (CADs) that influence the catalytic activities of different lysosomal enzymes. This action results in the side effect of phospholipidosis (PLD), which describes a detrimental increase in the phospholipid content in lysosomes. FIASMAs differ only slightly in their physico-chemical properties, but their effects on ASM activity and induction of the lysosomal phospholipid content vary significantly. In this study, we systematically induced minor chemical modifications to the FIASMAs imipramine, desipramine and fluoxetine. We generated a library of 45 new CADs with slightly different log P (logarithmic partition coefficient) and pKa (logarithmic acid dissociation constant) values. The effects of the compounds on the ASM activity and lysosomal phospholipid content were assessed in cell culture assays. We identified four compounds with beneficial effects, i.e., increased ASM activity inhibition and reduced PLD induction compared with the original drugs. The compounds HT04, RH272B and RH272D outperformed the original imipramine, whereas RH281A performed better than desipramine. Thus, minor chemical variations of CADs impact lysosomal metabolism in a specific manner and can lead to antidepressant drugs with less deleterious side effects.

Determination of acid sphingomyelinase activity in biological samples with ultra-performance liquid chromatographic assay

A simple, precise and accurate ultra-performance liquid chromatographic (UPLC) method was developed for the quantitative determination of the acid sphingomyelinase (ASM) activity in L-02 hepatocytes. The biological samples of cell homogenate were collected from L-02 hepatocytes and incubated with the fluorescent substrate BODIPY C12-Spm for 2 hr. The samples were determined using ACQUITY UPLC® BEH Amide chromatographic column with mobile phase containing acetonitrile and 13 mM sodium acetate (97:3, v/v) at a flow rate of 0.8 mL/min. The injection volume was 5 μL. The eluent was monitored using a fluorescence detector set to excitation and emission wavelengths of 435 nm and 525 nm, respectively. Fluorescent product B12-Cer in chromatography analytical can be effectively separated in 2 min, showing good linearity in the range of 6.25–300 ng/mL with the correlation coefficient of 0.9926. The mean ASM activity in L-02 hepatocytes with different sample pretreatment methods, lysis buffer, ultrasonic disruption, and freeze-thawing with liquid nitrogen was 3225.76 ± 323.63, 3330.98 ± 277.99 and 3355.05 ± 267.77 ng/mgprot/h, respectively. Thus we think the current method can be used to detect ASM activity in traces of biological samples.

Developing recombinant phospholipase D1 (rPLD1) toxoid from Iranian Hemiscorpius lepturus scorpion and its protective effects in BALB/c mice

Hemiscorpius lepturus (H. lepturus) is one of the most dangerous scorpions and the most medically important scorpion in Iran. The clinical signs of H. lepturus envenomation, including dermonecrosis, hematuria, renal failure and early death, are attributed to phospholipase D activity. This study was conducted to develop a novel recombinant phospholipase D1 (rPLD1) toxoid and investigate its immunogenicity and protective effects against the lethality of H. lepturus venom. The lethal protein recombinant phospholipase D1 was expressed from PLD H. lepturus venom gland. The rPLD1 toxin was converted into toxoid (the first toxoid of H. lepturus PLD) with a 0.25% concentration of formalin and stored for ten days at room temperature. In the toxicity test, the lethal activity of recombinant phospholipase D1 was fully inhibited. When it reached up to 3 times higher than the maximal effective concentration of the purified toxin (11.1 μg), rPLD1 toxoid was used. The sphingomyelinase activity was inhibited when up to 5.4 times of the LD100 of the purified toxin (20 μg), toxoid was used. It was then used to produce an antibody in BALB/c as an antigen and the mice were then challenged with rPLD1 toxin and the whole venom. The immunogenicity of rPLD1 toxoid was evaluated and the maximum titer of the raised antibodies was determined by ELISA assay. The optimum titer for anti-rPLD1 toxoid sera was obtained at the third intraperitoneal injection of rPLD1 toxoid, and a high titer was reached at the fourth injection in the mice. This toxoid increased the amount of antibodies and produced a protective antiserum against the whole venom of H. lepturus and rPLD1 toxin. The in-vivo test results showed that the mice were completely resistant against 200 times the LD100 of recombinant phospholipase D1 and the whole venom of H. lepturus. To conclude, rPLD1 can be used in toxoid form as an immunogen in the production of a new generation of neutralizing antibodies against the lethality and toxicity of H. lepturus whole venom.

Activation of neutral sphingomyelinase 2 by starvation induces cell-protective autophagy via an increase in Golgi-localized ceramide

Autophagy is essential for optimal cell function and survival, and the entire process accompanies membrane dynamics. Ceramides are produced by different enzymes at different cellular membrane sites and mediate differential signaling. However, it remains unclear which ceramide-producing pathways/enzymes participate in autophagy regulation under physiological conditions such as nutrient starvation, and what the underlying mechanisms are. In this study, we demonstrate that among ceramide-producing enzymes, neutral sphingomyelinase 2 (nSMase2) plays a key role in autophagy during nutrient starvation. nSMase2 was rapidly and stably activated upon starvation, and the enzymatic reaction in the Golgi apparatus facilitated autophagy through the activation of p38 MAPK and inhibition of mTOR. Moreover, nSMase2 played a protective role against cellular damage depending on autophagy. These findings suggest that nSMase2 is a novel regulator of autophagy and provide evidence that Golgi-localized ceramides participate in cytoprotective autophagy against starvation.

Reactive oxygen species mediates 50-Hz magnetic field-induced EGF receptor clustering via acid sphingomyelinase activation

Purpose: Exposure to extremely low frequency electromagnetic fields (ELF-EMFs) could elicit biological effects including carcinogenesis. However, the detailed mechanisms by which these ELF-EMFs interact with biological system are currently unclear. Previously, we found that a 50-Hz magnetic field (MF) exposure could induce epidermal growth factor receptor (EGFR) clustering and phosphorylation on cell membranes. In the present experiment, the possible roles of reactive oxygen species (ROS) in MF-induced EGFR clustering were investigated.Materials and methods: Human amnion epithelial (FL) cells were exposed to a 50-Hz MF with or without N-acetyl-l-cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC). EGFR clustering on cellular membrane surface was analyzed using confocal microscopy after indirect immunofluorescence staining. The intracellular ROS level and acid sphingomyelinase (ASMase) activity were detected using an ROS assay kit and an Amplex® Red Sphingomyelinase Assay Kit, respectively.Results: Results showed that exposure of FL cells to a 50-Hz MF at 0.4 mT for 15 min significantly enhanced the ROS level, induced EGFR clustering and increased ASMase activity. However, pretreatment with NAC or PDTC, the scavenger of ROS, not only counteracted the effects of a 50-Hz MF on ROS level and AMS activity, but also inhibited the EGFR clustering induced by MF exposure.Conclusions: The present and previous data suggest that ROS mediates the MF-induced EGFR clustering via ASMase activation.

P-96 - Sphingolipidomic analysis in acne vulgaris patient serum shows decreased C24-ceramide and increased ceramide-1-phosphate levels

This study aimed to identify circulating levels of neutral sphingomyelinase activity (N-SMase), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), C16-C24 sphingomyelin (SM), C16-C24 ceramide (CER) in acne vulgaris (AV) patients and controls. Serum was collected from AV patients and age, gender matched controls. Serum levels of C16-C24 SMs and C16-C24 CERs were determined by multiple reaction monitoring (MRM) method using ultrafast-liquid chromatography coupled with tandem mass spectrometry. Serum activity of N-SMase was assayed by standard kit methods, C1P and S1P levels were determined by enzyme-linked immunosorbent assay (ELISA). A significant increase was observed in serum levels of C16 SM in patients with AV compared to controls. Very-long-chain C24 CER was significantly decreased in AV patients compared to controls. A significant positive correlation was found between serum total cholesterol levels and all measured SMs and CERs in both the control and patient groups. Patients with AV had increased circulating levels of C16 SM, C1P and lower circulating levels of C24 CER compared to healthy controls, which may provide prognostic value for the disease. Future studies are needed to understand the role of altered sphingolipid levels in the pathophysiology of AV.