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.

Relationships between ocular surface sphingomyelinases, Meibum and Tear Sphingolipids, and clinical parameters of meibomian gland dysfunction.

PURPOSE: Sphingolipids (SPL) are a class of lipid molecules that play important functional and structural roles in our body and are a component of meibum. Sphingomyelinases (SMases) are key enzymes in sphingolipid metabolism that hydrolyze sphingomyelin (SM) and generate ceramide (Cer). The purpose of this study was to examine relationships between ocular surface SMases, SPL composition, and parameters of Meibomian gland dysfunction (MGD). METHODS: Individuals were grouped by meibum quality (n = 25 with poor-quality, MGD, and n = 25 with good-quality, control). Meibum and tears were analyzed with LC-MS to quantify SPL classes: Cer, Hexosyl-Ceramide (Hex-Cer), SM, Sphingosine (Sph), and sphingosine 1-phosphate (S1P). SMase activity in tears were quantified using a commercially available 'SMase assay'. Statistical analysis included multiple linear regression analyses to assess the impact of SMase activity on lipid composition, as well as ocular surface symptoms and signs of MGD. RESULTS: Demographic characteristics were similar between the two groups. nSMase and aSMase levels were lower in the poor vs good quality group. aSMase activity in tears negatively correlated with SM in meibum and tears and positively with Sph in meibum and S1P in tears. Lower SMase activity were associated with signs of MGD, most notably Meibomian gland dropout. CONCLUSION: This study suggests that individuals with MGD have reduced enzymatic activity of SMases in tears. Specifically, individuals with poor vs good meibum quality were noted to have alterations in SMase activity and SPL composition of meibum and tears which may reflect deviations from normal lipid metabolism in individuals with MGD.

Systemic Elevation of n-3 Polyunsaturated Fatty Acids (n-3-PUFA) Is Associated with Protection against Visual, Motor, and Emotional Deficits in Mice following Closed-Head Mild Traumatic Brain Injury.

Traumatic brain injury (TBI) causes neuroinflammation and neurodegeneration leading to various pathological complications such as motor and sensory (visual) deficits, cognitive impairment, and depression. N-3 polyunsaturated fatty acid (n-3 PUFA) containing lipids are known to be anti-inflammatory, whereas the sphingolipid, ceramide (Cer), is an inducer of neuroinflammation and degeneration. Using Fat1+-transgenic mice that contain elevated levels of systemic n-3 PUFA, we tested whether they are resistant to mild TBI-mediated sensory-motor and emotional deficits by subjecting Fat1-transgenic mice and their WT littermates to focal cranial air blast (50 psi) or sham blast (0 psi, control). We observed that visual function in WT mice was reduced significantly following TBI but not in Fat1+-blast animals. We also found Fat1+-blast mice were resistant to the decline in motor functions, depression, and fear-producing effects of blast, as well as the reduction in the area of oculomotor nucleus and increase in activated microglia in the optic tract in brain sections seen following blast in WT mice. Lipid and gene expression analyses confirmed an elevated level of the n-3 PUFA eicosapentaenoic acid (EPA) in the plasma and brain, blocking of TBI-mediated increase of Cer in the brain, and decrease in TBI-mediated induction of Cer biosynthetic and inflammatory gene expression in the brain of the Fat1+ mice. Our results demonstrate that suppression of ceramide biosynthesis and inflammatory factors in Fat1+-transgenic mice is associated with significant protection against the visual, motor, and emotional deficits caused by mild TBI. This study suggests that n-3 PUFA (especially, EPA) has a promising therapeutic role in preventing neurodegeneration after TBI.

Synthesis and characterization of a new two photon excitable acid sphingomyelinase FRET probe.

Recently, FRET probes for acid sphingomyelinase (ASM) have enabled the observation of enzyme activity in intact cells for the first time. Here we present an ASM FRET probe specifically optimized for 2-photon excitation. To facilitate probe characterization and comparison to the previous probe, we mixed the two intact probes with defined amounts of the probes' ceramide cleavage products and mounted them on lipid beads. Directly excited NBD FRET acceptor fluorescene proved to be a useful means of reference and showed that the new probe is brighter, albeit only moderately, than the previous one. The new probe was then used to detect inhibition by various ASM inhibitors microscopically for the first time. Also in cells, directly excited acceptor fluorescence proved to be a useful parameter in addition to FRET to visualize inhibition of ASM.

Intracellular Wireless Analysis of Single Cells by Bipolar Electrochemiluminescence Confined in a Nanopipette.

The inside walls of a nanopipette tip are decorated by a Pt deposit that is used as an open bipolar electrochemiluminescence (ECL) device to achieve intracellular wireless electroanalysis. The synergetic actions of nanopipette and of bipolar ECL lead to the spatial confinement of the voltage drop at the level of the Pt deposit, which generates ECL emission from luminol. The porous structure of Pt deposit permits the electrochemical transport of intracellular molecules into the nanopipette that is coupled with enzymatic reactions. Thus, the intracellular concentrations of hydrogen peroxide or glucose are measured in vivo as well as the intracellular sphingomyelinase activity. In comparison with the classic bipolar ECL, the remarkably low potential applied in our approach is restricted inside the nanopipette and it minimizes the potential bias of the voltage on the cellular activity. Accordingly, this wireless ECL approach provides a new direction for analysis of single living cells.

FRET probes for measuring sphingolipid metabolizing enzyme activity.

Förster resonance energy transfer (FRET) probes are unique tools in biology, as they allow for a non-destructive monitoring of a certain state of a biomolecule or of an artificial substrate within living cells in real time. FRET substrates indicate their relative cleavage rate and thus the in situ activity of a given enzyme. In contrast to quenched probes or turn-on probes, one of the two separate signals of the FRET probes can be used as internal reference, which makes ratio-imaging and quantitation of cleavage events independent of cellular delivery possible. In this review, we describe the first examples of sphingolipid FRET probes in comparison to different alternative probes. Finally, we give an outlook on future probes and their potential application.

A Robust Liposomal Platform for Direct Colorimetric Detection of Sphingomyelinase Enzyme and Inhibitors.

The enzyme sphingomyelinase (SMase) is an important biomarker for several diseases such as Niemann Pick's, atherosclerosis, multiple sclerosis, and HIV. We present a two-component colorimetric SMase activity assay that is more sensitive and much faster than currently available commercial assays. Herein, SMase-triggered release of cysteine from a sphingomyelin (SM)-based liposome formulation with 60 mol % cholesterol causes gold nanoparticle (AuNP) aggregation, enabling colorimetric detection of SMase activities as low as 0.02 mU/mL, corresponding to 1.4 pM concentration. While the lipid composition offers a stable, nonleaky liposome platform with minimal background signal, high specificity toward SMase avoids cross-reactivity of other similar phospholipases. Notably, use of an SM-based liposome formulation accurately mimics the natural in vivo substrate: the cell membrane. We studied the physical rearrangement process of the lipid membrane during SMase-mediated hydrolysis of SM to ceramide using small- and wide-angle X-ray scattering. A change in lipid phase from a liquid to gel state bilayer with increasing concentration of ceramide accounts for the observed increase in membrane permeability and consequent release of encapsulated cysteine. We further demonstrated the effectiveness of the sensor in colorimetric screening of small-molecule drug candidates, paving the way for the identification of novel SMase inhibitors in minutes. Taken together, the simplicity, speed, sensitivity, and naked-eye readout of this assay offer huge potential in point-of-care diagnostics and high-throughput drug screening.

Amplification of a FRET Probe by Lipid-Water Partition for the Detection of Acid Sphingomyelinase in Live Cells.

Real-time monitoring of acid sphingomyelinase (ASM) activity is crucial for investigating its role in lipid-mediated signaling processes. In this study, we synthesized fluorescent phosphosphingolipids capable of FRET by phosphorodichloridate chemistry. These sphingomyelin analogues are substrates for recombinant human ASM and can be used to monitor ASM activity by fluorescence spectroscopy. Incubation with cell lysates from wild-type and knock-out mice further confirmed probe cleavage to be exclusive to ASM. We also systematically exploited the environmental sensitivity of the fluorophores to achieve significant increases in responsiveness. This concept may be transferred to other lipid probes in the future. The ASM activity in live cells was imaged by two-photon-excitation microscopy.

Mouse Thyroid Gland Changes in Aging: Implication of Galectin-3 and Sphingomyelinase.

Prevalence of thyroid dysfunction and its impact on cognition in older people has been demonstrated, but many points remain unclarified. In order to study the effect of aging on the thyroid gland, we compared the thyroid gland of very old mice with that of younger ones. We have first investigated the changes of thyroid microstructure and the possibility that molecules involved in thyroid function might be associated with structural changes. Results from this study indicate changes in the height of the thyrocytes and in the amplitude of interfollicular spaces, anomalous expression/localization of thyrotropin, thyrotropin receptor, and thyroglobulin aging. Thyrotropin and thyrotropin receptor are upregulated and are distributed inside the colloid while thyroglobulin fills the interfollicular spaces. In an approach aimed at defining the behavior of molecules that change in different physiopathological conditions of thyroid, such as galectin-3 and sphingomyelinase, we then wondered what was their behavior in the thyroid gland in aging. Importantly, in comparison with the thyroid of young animals, we have found a higher expression of galectin-3 and a delocalization of neutral sphingomyelinase in the thyroid of old animals. A possible relationship between galectin-3, neutral sphingomyelinase, and aging has been discussed.

Synthesis of substrates for periodate-coupled assay of phospholipases C and sphingomyelinases.

A series of 4-nitrophenyl (pNP) and 4-methylumbelliferyl (4MU) substrate analogues of phosphatidyl choline (PC) and phosphatidic acid (PA) were synthesized from 4-bromo-1-butene by ether formation, olefin epoxidation and ring opening with the phosphate head group. The pNP PC analogue, 4-(4-nitrophenoxy)-2-hydroxy-butyl-1-phosphoryl choline (1) was evaluated in assays of fungal sphingomyelinases, also displaying phospholipase C activity. Reactions were terminated with a periodate-containing stop solution, leading to liberation of pNP, quantified spectrophotometrically in an end-point measurement. A kinetic evaluation of sphingomyelinases from Kionochaeta sp. and Penicillium emersonii showed relatively high KM and low kcat values for this substrate, limiting its practical applicability in assays with low sphingomyelinase concentrations.

Acid sphingomyelinase, a novel negative biomarker of ovarian cancer.

OBJECTIVE: Ovarian cancer is the sixth most common cancer and the main cause of death in women. However, the molecular mechanism for the cause of the ovarian cancer has not been fully elucidated. Acid sphingomyelinase (ASM), a lipid hydrolase, has been suggested for treating cancer and may affect the development of ovarian cancer. We want to find the function of ASM in the development of ovarian cancer. PATIENTS AND METHODS: Human ovarian cancer cells HO 8910 (HOCC) and human primary ovarian cells (HPOC) were transfected with ASM gene and ASM RNAi. Real-time qPCR and western blot analysis was carried out to examine the level of ASM. The growth rate of transfected and non-transfected cells was measured. Ovarian biopsies were collected from 80 ovarian cancer patients and 20 healthy subjects. RESULTS: The growth rate of HOCC and HPOC was decreased by 22% and 19% in the ASM-transfected group compared with non-transfected group. Inversely, the growth rate of HOCC and HPOC was increased by 16% and 35% in the ASM-RNAi-transfected group compared with non-transfected group. In the transfected and non-transfected cells, the change level of SAM was approved by Real-time qPCR and western blot analysis. The levels of SAM were reducing with the development of ovarian cancer. CONCLUSIONS: SAM is higher expressed in normal cell than that in ovarian cancer, and can be a negative biomarker for the diagnosis of ovarian cancer. SAM can be developed a new drug for the ovarian cancer therapy.

Diaphragm dysfunction in heart failure is accompanied by increases in neutral sphingomyelinase activity and ceramide content.

AIMS: Chronic heart failure (CHF) causes inspiratory (diaphragm) muscle weakness and fatigue that contributes to dyspnoea and limited physical capacity in patients. However, the mechanisms that lead to diaphragm dysfunction in CHF remain poorly understood. Cytokines and angiotensin II are elevated in CHF and stimulate the activity of the enzyme sphingomyelinase (SMase) and accumulation of its reaction product ceramide. In the diaphragm, SMase or ceramide exposure in vitro causes weakness and fatigue. Thus, elevated SMase activity and ceramide content have been proposed as mediators of diaphragm dysfunction in CHF. In the present study, we tested the hypotheses that diaphragm dysfunction was accompanied by increases in diaphragm SMase activity and ceramide content. METHODS AND RESULTS: Myocardial infarction was used to induce CHF in rats. We measured diaphragm isometric force, SMase activity by high-performance liquid chromatography, and ceramide subspecies and total ceramide using mass spectrometry. Diaphragm force was depressed and fatigue accelerated by CHF. Diaphragm neutral SMase activity was increased by 20% in CHF, while acid SMase activity was unchanged. We also found that CHF increased the content of C18 -, C20 -, and C24 -ceramide subspecies and total ceramide. Downstream of ceramide degradation, diaphragm sphingosine was unchanged, and sphingosine-1-phosphate level was increased in CHF. CONCLUSION: Our major novel finding was that diaphragm dysfunction in CHF rats was accompanied by higher diaphragm neutral SMase activity, which is expected to cause the observed increase in diaphragm ceramide content.

[Sphingolipids in skeletal muscles of C57B1/6 mice after short-term simulated microgravity].

For the first time in skeletal muscle, the sphingolipid profile and key enzymes involved in the generation of ceramide in cells were investigated in simulated microgravity. It was found that, in C57B1/6 mice, the 4-day hindlimb unloading, in addition to reducing the mass of m. soleus, leads to the ceramide accumulation (3-fold) and the decrease of sphingomyelin content in this muscle (7.2-fold), as well as to the increase (2.7-fold) of protein level of acid sphingomyelinase. In a loaded m. biceps brachii the amount of ceramide is also enhanced, but both the amount of sphingomyelin and sphingomyelinase, as well as the muscle mass do not change, while the level of serine palmitoyltranspherase becomes significantly lower than in control mice. Taking into account the negative effects of ceramide in skeletal muscle (insulin resistance, inhibition of protein synthesis and increase of its decay) we can assume that sphingolipid mechanisms may be involved in the development of structural and functional abnormalities of skeletal muscle under conditions of weightlessness.

Analysis of acid sphingomyelinase activity in dried blood spots using tandem mass spectrometry.

BACKGROUND: Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS) method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening. METHODS: ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6-sphingomyelin (C(29)H(59)N(2)O(6)P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C(22)H(43)NO(3))]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product). RESULTS: ASM activities were stable for up to 2 months at or below 4℃. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intra- and inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 µmol/h/L (mean 10.6) with a SD of 5.06 µmol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 µmol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 µmol/h/L). CONCLUSIONS: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP.

Oxidative stress triggers Ca-dependent lysosome trafficking and activation of acid sphingomyelinase.

Recent studies demonstrate that rapid translocation of the acid sphingomyelinase (ASM), a lysosomal hydrolase, to the outer leaflet of the cell membrane and concomitant release of ceramide constitute a common cellular signaling cascade to various stimuli including CD95 ligation, UV-irradiation, bacterial and viral infections. Reactive oxygen species (ROS) were shown to play a crucial role in regulating this signaling cascade at least for some bacterial infections and UV-irradiation. However, the precise role of ROS for regulation of ASM is unknown. Here, by confocal microscopy and flow cytometry analysis, we demonstrate that hydrogen peroxide (H(2)O(2)), a primary form of ROS in mammalian cells, induces very rapid translocation of ASM and formation of ceramide-enriched membrane platforms in the plasma membrane of Jurkat T cells. In parallel, H(2)O(2) triggers lysosome trafficking and fusion with the plasma membrane, i.e. lysosome exocytosis, as detected by exposure of a lysosome-associated protein, LAMP1. Depletion of intracellular Ca(2+) by cell permeable EGTA-AM inhibits H(2)O(2)-induced lysosome exocytosis, ASM translocation and formation of ceramide-enriched platforms. Pharmacological inhibition or genetic deficiency of ASM did not affect H(2)O(2)-induced lysosome exocytosis. These results indicate that ROS-induced membrane translocation of ASM is mediated by exocytosis of lysosomes, which is dependent on intracellular Ca(2+) release.

Study of Staphylococcus aureus collected at slaughter from dairy cows with chronic mastitis.

Staphylococcus aureus is one of the most important pathogens associated with bovine mastitis. Recent studies have shown that Staph. aureus strains may differ in virulence, and in their ability to disseminate across commercial dairy herds. The goal of this study was to determine whether Staph. aureus isolates differed in their ability to colonize mammary tissue, and whether such differences could be related to molecular characteristics. Quarter milk and mammary tissues of 22 cows from two dairy herds, were collected at slaughter and bacteriological analysis was performed. All Staph. aureus isolates were characterized by Pulsed Field Gel Electrophoresis (PFGE) and microarray. Overall 45 mammary quarters were infected and 20 Staph. aureus isolates were identified. The bacteria were mostly recovered from both milk and tissue of the same quarter in significantly higher numbers from herd A cows compared with herd B. Molecular characterization of the isolates showed distinct PFGE profiles for isolates from each herd. Differences in virulence factors between herds A and B isolates were evidenced The genes for enterotoxin D, J and R were present in herd A, those for G, I, N, M, O and U were shown in herd B, whilst both components of the leukocidin lukD/E genes were only carried by herd A isolates. Furthermore, all herd A isolates showed ß-haemolysin activity, which was absent in all but one isolate from herd B. Therefore our data indicate that Staph. aureus isolates showing differences in their ability to disseminate and colonize across quarters, also have significantly different virulence characteristics.

5,7,3'-Trihydroxy-3,4'-dimethoxyflavone inhibits the tubulin polymerization and activates the sphingomyelin pathway.

Flavonoids are polyphenolic compounds which display a vast array of biological activities and are among the most promising anti-cancer agents. The derivative of quercetin, 5,7,3'-trihydroxy-3,4'-dimethoxyflavone (THDF), is a natural flavonoid that inhibits cell proliferation and induces apoptosis in human leukemia cells. Here we show that THDF induces cell-cycle arrest in the M phase and inhibits tubulin polymerization. This was associated with the accumulation of cyclin B1 and p21(Cip1) , changes in the phosphorylation status of cyclin B1, Cdk1, Cdc25C, and MPM-2, and activation of the acidic sphingomyelinase (ASMase). Moreover, desipramine attenuated THDF-mediated cell death, indicating a crucial role of ASMase in the mechanism of cell death. In vivo studies on the athymic nude mouse xenograft model also confirmed that THDF inhibits growth of human leukemia cells and suggest that this compound may have therapeutic value.

Aerobic training in rats increases skeletal muscle sphingomyelinase and serine palmitoyltransferase activity, while decreasing ceramidase activity.

Sphingolipids are important components of cell membranes that may also serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. The aim of this study was to examine the effect of 5 weeks of aerobic training on key enzymes and intermediates of ceramide metabolism in skeletal muscles. The experiments were carried out on rats divided into two groups: (1) sedentary and (2) trained for 5 weeks (on a treadmill). The activity of serine palmitoyltransferase (SPT), neutral and acid sphingomyelinase (nSMase and aSMase), neutral and alkaline ceramidases (nCDase and alCDase) and the content of sphingolipids was determined in three types of skeletal muscle. We also measured the fasting plasma insulin and glucose concentration for calculating HOMA-IR (homeostasis model assessment) for estimating insulin resistance. We found that the activities of aSMase and SPT increase in muscle in the trained group. These changes were followed by elevation in the content of sphinganine. The activities of both isoforms of ceramidase were reduced in muscle in the trained group. Although the activities of SPT and SMases increased and the activity of CDases decreased, the ceramide content did not change in any of the studied muscle. Although ceramide level did not change, we noticed increased insulin sensitivity in trained animals. It is concluded that training affects the activity of key enzymes of ceramide metabolism but also activates other metabolic pathways which affect ceramide metabolism in skeletal muscles.