Sphingolipid Profiles Research Articles

P0019 Investigation of the serum sphingolipid and ceramide profile in patients with Crohn's disease at different stages of the disease

Abstract Background Ceramides and sphingolipids are components of the cell membrane of the intestinal epithelium and contribute to the integrity of the intestinal barrier (1, 2). The expression of specific sphingolipid and ceramide subgroups can vary depending on the inflammatory activity of Inflammatory bowel diseases (IBD) and may influence the course of the disease. A distinct lipid profile was shown for ulcerative colitis compared to healthy subjects and different expression depending on disease activity. Less data is available for Crohn's disease (CD) (3). In this study, patients with MC at different stages of the disease were to be analysed with regard to their sphingolipid and ceramide profile using liquid chromatography tandem mass spectrometry (LC-MS/MS). The aim was to identify biomarkers that correlate with inflammatory activity so that they can be used in diagnostics and therapeutic monitoring. They could also serve as a target for a new therapeutic approach. Methods Patients who were experienced in therapy and had relevant disease activity at the time of blood sampling were included in the study after receiving a positive ethics vote. Patients were considered as treatment-experienced if they had received at least 2 prior biologic therapies. Disease activity was defined by clinical activity indices (Harvey-Bradshaw index >/= 4) and faecal calprotectin (>/= 250 ug/g). Further inclusion criteria were: Age of majority, a >3 month diagnosis of Crohn's disease and written informed consent. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to quantify the sphingolipids in plasma. The analytes were extracted using liquid-liquid extraction and measured using two different LC-MS/MS methods for sphingolipids and ceramides. Results In the comparison between the Crohn's disease and the control cohort, there were significant differences for the ceramides Cer d18:1/16:0 (p=0.009), Cer d 18.1/18:0 (p=0.025) and Cer 18.1/24:0 (p=0.035) showed higher concentrations in the MC group. For the lactosylceramides, there were significant group differences for LacCer 18:1/16:0 (p=0.005) and LacCer 18.1/18:0 (p=0.009). For sphingosine-1-phosphatase, there were significant differences for S1P 16:0 (p<0.001). LacCer and S1P concentrations were also higher in the MC group. Conclusion In the present study, a distinct difference in individual sphingolipid and ceramide concentrations could be determined in patients with Crohn's disease under an advanced therapy line and relevant disease activity compared to a control group. Overall, the concentrations in the MC group were higher in the significantly different parameters. Overall, the data on the role of sphingolipids and ceramides in IBD is not yet conclusive and further research is warranted.

Altered sphingolipid profile in response to skeletal muscle injury in a mouse model of type 1 diabetes mellitus.

A complication of type 1 diabetes mellitus (T1DM) is diabetic myopathy that includes reduced regenerative capacity of skeletal muscle. Sphingolipids are a diverse family of lipids with roles in skeletal muscle regeneration. Some studies have found changes in sphingolipid species levels in T1DM, however, the effect of T1DM on a sphingolipid panel in regenerating skeletal muscle has not been examined. Wild-type (WT) and diabetic Ins2Akita+/- (Akita) mice received cardiotoxin-induced muscle injury in their left quadriceps, gastrocnemius-plantaris-soleus, and tibialis anterior muscles with the contralateral muscles serving as uninjured controls. Muscles were collected at 1, 3, 5, or 7 days postinjury. In regenerating muscle from Akita mice, lipid staining with BODIPY 493/503 revealed increased intramyocellular and total lipids and perilipin-1-positive cell numbers as compared with WT. Liquid chromatography-mass spectrometry of quadriceps was used to identify sphingolipid levels in skeletal muscle. The C22:0 and C24:0 ceramides were significantly elevated in uninjured Akita, whereas ceramide C24:1 was decreased in injured Akita compared with WT. Ceramide-1-phosphate was increased in Akita compared with WT regardless of injury, whereas sphingosine-1-phosphate (S1P) was elevated with injury in WT but this response was muted in Akita mice. Western blotting of key enzymes involved in sphingolipid metabolism revealed S1P lyase, the enzyme that degrades S1P irreversibly, was significantly elevated in the injured muscle in Akita mice during regeneration, in accordance with lower S1P levels. This mouse model of T1DM demonstrates sphingolipidomic changes that may contribute to delayed muscle regeneration.NEW & NOTEWORTHY Muscle lipids become elevated, and the sphingolipid profile is altered by T1DM in skeletal muscle regeneration. A loss of S1P is accompanied by greater expression of sphingosine-1-phosphate lyase (SPL) in response to injury in Akita mice, suggesting a role for sphingolipids in the attenuated repair of skeletal muscle in T1DM rodent models. Although ceramide-1-phosphate (C1P) is increased with T1DM, there was no increase in ceramide kinase (CerK) suggesting an alternative route of ceramide phosphorylation in skeletal muscle.

Sphingolipid profiling as a biomarker of type 2 diabetes risk: evidence from the MIDUS and PREDIMED studies

BackgroundType 2 diabetes (T2D) has become a worldwide pandemic. While ceramides may serve as intermediary between obesity-related lipotoxicity and T2D, the relationship with simple glycosphingolipids remains uncertain. The aim of this study was to characterize the associations between blood glycosphingolipid and ceramide species with T2D and to identify a circulating sphingolipid profile that could serve as novel biomarker for T2D risk.MethodsCross-sectional relationship between sphingolipid levels, insulin resistance, and T2D prevalence were evaluated in 2,072 American adults from MIDUS cohort. Prospectively, the association between sphingolipid species and the incidence of T2D was analyzed using a case-cohort design nested within the PREDIMED trial (250 cases and a random sample of 692 participants, with 3.8 years of median follow-up). Circulating levels of sphingolipid species in both populations were measured using LC/MS. Hazard ratios were estimated with weighted Cox regression models using Barlow weights.ResultsIn American adults, only CER18:0 and CER22:0 were linked to insulin resistance and a higher prevalence of T2D. Conversely, three lactosylceramides (LCER 14:0, 16:0, and 24:1) showed a strong inverse relationship with both insulin resistance and T2D. These findings led to development of two sphingolipid scores. In the prospective analysis, these scores consistently predicted a reduced risk of T2D incidence in PREDIMED (HR: 0.64, 95% CI 0.44 to 0.94 and 0.58, 0.40 to 0.85 respectively) between extreme quartiles, with 5-year absolute risk differences of 9.6% (95% CI: 0.3–20.5%) and 11.4% (1.0–21.6%). They were validated in the same trial with samples obtained after 1 year of follow-up.ConclusionsOur findings support the potential usefulness of circulating sphingolipid profiles as novel biomarkers for T2D risk. Moreover, this study opens the door for future research on the predictive value and possible protective roles of lactosylceramides in T2D.Graphical abstract

Inhibiting De Novo Biosynthesis of Ceramide by L-Cycloserine Can Prevent Light-Induced Retinal Degeneration in Albino BALB/c Mice.

Retinal degenerative diseases lead to irreversible vision loss due to photoreceptor cell death, driven by complex genetic and environmental factors. Ceramide, a sphingolipid metabolite, emerges as a critical mediator in the apoptotic cascade associated with retinal degeneration. Our previous work demonstrated L-Cycloserine's ability to protect photoreceptor-derived cells from oxidative stress by inhibiting the de novo ceramide pathway and thus prompting further investigation on its effect in the in vivo retina. This study investigates the potential of L-Cycloserine to protect albino BALB/c mice against light-induced retinal degeneration (LIRD). L-Cycloserine, in an optimal dose, administered systemically 30 min before LIRD, was found to prevent photoreceptor cell death significantly from light-induced degeneration. We further determined the retinal bioavailability and pharmacokinetic behavior of L-Cycloserine, its effect on sphingolipid profile, expression of sphingolipid biosynthetic, and cell death-promoting genes and proteins from the retina to understand the underlying mechanisms. This study lays the groundwork for further preclinical and clinical investigations into L-Cycloserine's potential as a novel therapeutic in treating retinal degenerative diseases.

Monitoring concentration and lipid signature of plasma extracellular vesicles from HR+ metastatic breast cancer patients under CDK4/6 inhibitors treatment.

Extracellular vesicles (EVs) are cell-derived small membrane structures that transport various molecules. They have emerged as potential circulating biomarkers for monitoring responses to cancer therapies. This study aimed to comprehensively characterize plasma-carried EVs in hormone receptor-positive (HR+) metastatic breast cancer (MBC) patients treated with first-line CDK4/6 inhibitors (iCDK4/6) combined with endocrine therapy. MBC patients were classified into three groups based on their response to therapy: resistant, intermediate or sensitive. In a prospective cohort, we monitored the concentration of circulating EVs, analyzed their lipid signature and correlated these factors with treatment response. To facilitate the translation of EV research to clinical practice, we established a three-step procedure: (1) EVs were isolated from plasma using semi-automatized size exclusion chromatography (SEC); (2) EV concentration, termed vesiclemia, was determined by drop counting via interferometric light microscopy (ILM); and (3) EV lipid composition was analyzed by mass spectrometry. ILM-based vesiclemia values were highly fluctuating upon iCDK4/6 treatment, while early increase associated with accelerated progression. Of note, vesiclemia remained a steady parameter over a 1-year period in age-matched healthy women. Additionally, analysis of the EV cargo unveiled a distinct sphingolipid profile, characterized by increased levels of ceramides and sphingomyelins in resistant patients within the first 2 months of treatment. Based on 16 sphingolipid species, sensitive and resistant patients were correctly classified with an overall accuracy of 82%. This specific sphingolipid pattern was exclusively discernible within EVs, and not in plasma, highlighting the significance of EVs in the early prediction of individual responses to iCDK4/6 and disease progression. Overall, this study provides insights of the longitudinal characterization of plasma-borne EVs in both a healthy group and HR+ MBC patients under iCDK4/6 therapies. Combined vesiclemia and EV sphingolipid profile emphasize the promising potential of EVs as non-invasive biomarkers for monitoring early treatment response.

Targeting an altered sphingolipid profile with consecutive lipotoxicity as therapeutic approach to calcific aortic valve disease

Abstract Background Aortic stenosis due to calcific aortic valve disease (CAVD) is the most common valvular heart disease. In symptomatic patients, without repair the prognosis is poor. Treatment to date is based on surgery or catheter-based interventions. Pharmacological therapy options to prevent the progression of valve calcification are not available. In vascular smooth muscle cells, cellular accumulation of sphingolipids is associated with a proinflammatory response in the sense of lipotoxicity. Chronic inflammatory processes are indeed also essential for aortic valve calcification. Purpose With the subsequent goal of developing innovative treatment approaches for CAVD, we therefore aim to elucidate the unclear role of sphingolipids in the development of CAVD. Methods Human aortic valve and plasma samples of CAVD patients and controls (n=26) were analysed by liquid chromatography–mass spectrometry for lipidomics and proteomics. Human aortic valve interstitial cells (hVICs) were stimulated with ceramides. Calcification was detected by alizarin red staining. mRNA expression of bone-related proteins was determined by qPCR. NF-kB pathway was assessed by proteome profiler. Myriocin and GW4869 were applied to inhibit ceramide biosynthesis. PDTC and MCC950 were used to inhibit NF-kB Pathway and NLRP3 activation. ApoE-/- mice received a gain-of-function PCSK9 adeno-associated virus vector and fed high cholesterol diet for 14 weeks. Myriocin was applied orally over the treatment period to inhibit ceramide de novo synthesis in vivo. Murine echocardiography was used to measure transvalvular velocity and left ventricular function. Calcification degree of murine aortic valve was determined by histological staining. Results A significantly altered sphingolipid profile was observed in aortic valve tissue of CAVD patients (Fig. 1). Ceramide species e.g. Cer (17:1;2O/16:0) were increased. Verifying the biological relevance of these findings in vitro, C2-Cer (d18:1/2:0) enhanced hVIC calcification (Fig. 2). Accordingly, ALP activity and mRNA expression of osteogenic genes RUNX2, ALPL and MSX2 were increased. Furthermore, C2-Cer (d18:1/2:0) enhanced NF-kB p65 phosphorylation and NLRP3 activation, while treatment with both PDTC and MCC950 protected against C2-Cer (d18:1/2:0) induced calcification. Supporting the role of sphingolipid biosynthesis in hVIC calcification, Myriocin and GW4869 both reduced ox-LDL-induced VIC calcification which was reversed by C2-Cer(d18:1/2:0). Finally, Myriocin reduced the degree of calcification and transvalvular jet velocity of aortic valve in the PCSK9-AAV ApoE-/- mice model. Conclusion CAVD is accompanied by an accumulation of ceramides causing lipotoxicity in human aortic valve tissue. Pharmacological modulation of sphingolipid metabolism is an effective approach to prevent the development and progression of aortic valve calcification in vivo and should be considered as a future therapeutic strategy in CAVD patients.Volcano Plot of CAVD tissue lipidomicsC2 Ceramide enhances hVIC calcification

Unraveling the glycosphingolipid metabolism by leveraging transcriptome-weighted network analysis on neuroblastic tumors

BackgroundGlycosphingolipids (GSLs) are membrane lipids composed of a ceramide backbone linked to a glycan moiety. Ganglioside biosynthesis is a part of the GSL metabolism, which involves sequential reactions catalyzed by specific enzymes that in part have a poor substrate specificity. GSLs are deregulated in cancer, thus playing a role as potential biomarkers for personalized therapy or subtype classification. However, the analysis of GSL profiles is complex and requires dedicated technologies, that are currently not included in the commonly utilized high-throughput assays adopted in contexts such as molecular tumor boards.MethodsIn this study, we developed a method to discriminate the enzyme activity among the four series of the ganglioside metabolism pathway by incorporating transcriptome data and topological information of the metabolic network. We introduced three adjustment options for reaction activity scores (RAS) and demonstrated their application in both exploratory and comparative analyses by applying the method on neuroblastic tumors (NTs), encompassing neuroblastoma (NB), ganglioneuroblastoma (GNB), and ganglioneuroma (GN). Furthermore, we interpreted the results in the context of earlier published GSL measurements in the same tumors.ResultsBy adjusting RAS values using a weighting scheme based on network topology and transition probabilities (TPs), the individual series of ganglioside metabolism can be differentiated, enabling a refined analysis of the GSL profile in NT entities. Notably, the adjustment method we propose reveals the differential engagement of the ganglioside series between NB and GNB. Moreover, MYCN gene expression, a well-known prognostic marker in NTs, appears to correlate with the expression of therapeutically relevant gangliosides, such as GD2. Using unsupervised learning, we identified subclusters within NB based on altered GSL metabolism.ConclusionOur study demonstrates the utility of adjusting RAS values in discriminating ganglioside metabolism subtypes, highlighting the potential for identifying novel cancer subgroups based on sphingolipid profiles. These findings contribute to a better understanding of ganglioside dysregulation in NT and may have implications for stratification and targeted therapeutic strategies in these tumors and other tumor entities with a deregulated GSL metabolism.

Sphingolipid Profiles in Small Extracellular Vesicle of Placental Origin as a Potential Indicator of Adverse Pregnancy Outcomes in Gestational Diabetes Mellitus

Sphingolipid Profiles in Small Extracellular Vesicle of Placental Origin as a Potential Indicator of Adverse Pregnancy Outcomes in Gestational Diabetes Mellitus

The sphingosine kinase 2 inhibitors ABC294640 and K145 elevate (dihydro)sphingosine 1-phosphate levels in various cells

Sphingosine kinases (SphKs), enzymes that produce the bioactive lipids dihydrosphingosine 1-phosphate (dhS1P) and sphingosine 1-phosphate (S1P), are associated with various diseases, including cancer and infections. For this reason, a number of SphK inhibitors have been developed. Although off-target effects have been described for selected agents, SphK inhibitors are mostly used in research without monitoring the effects on the sphingolipidome. We have now investigated the effects of seven commonly used SphK inhibitors (5c, ABC294640 (opaganib), N,N-dimethylsphingosine, K145, PF-543, SLM6031434, and SKI-II) on profiles of selected sphingolipids in Chang, HepG2, and human umbilical vein endothelial cells. While we observed the expected (dh)S1P reduction for N,N-dimethylsphingosine, PF-543, SKI-II, and SLM6031434, 5c showed hardly any effect. Remarkably, for K145 and ABC294640, both reported to be specific for SphK2, we observed dose-dependent strong increases in dhS1P and S1P across cell lines. Compensatory effects of SphK1 could be excluded, as this observation was also made in SphK1-deficient HK-2 cells. Furthermore, we observed effects on dihydroceramide desaturase activity for all inhibitors tested, as has been previously noted for ABC294640 and SKI-II. In additional mechanistic studies, we investigated the massive increase of dhS1P and S1P after short-term cell treatment with ABC294640 and K145 in more detail. We found that both compounds affect sphingolipid de novo synthesis, with 3-ketodihydrosphingosine reductase and dihydroceramide desaturase as their targets. Our study indicates that none of the seven SphK inhibitors tested was free of unexpected on-target and/or off-target effects. Therefore, it is important to monitor cellular sphingolipid profiles when SphK inhibitors are used in mechanistic studies.

Sphingolipid changes in mouse brain and plasma after mild traumatic brain injury at the acute phases

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

520-P: Plasma Sphingolipid Profiles Correlate with Circulating Lipoproteins and Adiposity

520-P: Plasma Sphingolipid Profiles Correlate with Circulating Lipoproteins and Adiposity

Acid ceramidase expression reduces IFNγ secretion by mouse CD4+ T cells and is crucial for maintaining B-cell numbers in mice.

Acid ceramidase (Ac) is a lysosomal enzyme catalyzing the generation of sphingosine from ceramide, and Ac inhibitors are currently being investigated as potential cancer therapeutics. Yet, the role of the Ac in immune responses, particularly anti-viral immunity, is not fully understood. To investigate the impact of Ac expression on various leukocyte populations, we generated a tamoxifen-inducible global knockout mouse model for the Ac (iAc-KO). Following tamoxifen administration to healthy mice, we extracted primary and secondary lymphoid organs from iAc-KO and wild-type (wt) littermates and subsequently performed extensive flow cytometric marker analysis. In addition, we isolated CD4+ T cells from the spleen and lymph nodes for sphingolipid profiling and restimulated them in vitro with Dynabeads™ Mouse T-activator CD3/CD28. Intracellular cytokine expression (FACS staining) was analyzed and secreted cytokines detected in supernatants. To study cell-intrinsic effects, we established an in vitro model for iAc-KO in isolated CD4+ T and B cells. For CD4+ T cells of iAc-KO versus wt mice, we observed reduced Ac activity, an increased ceramide level, and enhanced secretion of IFNγ upon CD3/CD28 costimulation. Moreover, there was a marked reduction in B cell and plasma cell and blast numbers in iAc-KO compared to wt mice. To study cell-intrinsic effects and in line with the 3R principles, we established in vitro cell culture systems for iAc-KO in isolated B and CD4+ T cells. Our findings pinpoint to a key role of the Ac in mature B and antibody-secreting cells and in IFNγ secretion by CD4+ T cells.

Effect of an eight-week high-intensity interval training programme on circulating sphingolipid levels in middle-aged adults at elevated cardiometabolic risk (SphingoFIT)-Protocol for a randomised controlled exercise trial.

Evidence indicates that sphingolipid accumulation drives complex molecular alterations promoting cardiometabolic diseases. Clinically, it was shown that sphingolipids predict cardiometabolic risk independently of and beyond traditional biomarkers such as low-density lipoprotein cholesterol. To date, little is known about therapeutic modalities to lower sphingolipid levels. Exercise, a powerful means to prevent and treat cardiometabolic diseases, is a promising modality to mitigate sphingolipid levels in a cost-effective, safe, and patient-empowering manner. This randomised controlled trial will explore whether and to what extent an 8-week fitness-enhancing training programme can lower serum sphingolipid levels of middle-aged adults at elevated cardiometabolic risk (n = 98, 50% females). The exercise intervention will consist of supervised high-intensity interval training (three sessions weekly), while the control group will receive physical activity counselling based on current guidelines. Blood will be sampled early in the morning in a fasted state before and after the 8-week programme. Participants will be provided with individualised, pre-packaged meals for the two days preceding blood sampling to minimise potential confounding. An 'omic-scale sphingolipid profiling, using high-coverage reversed-phase liquid chromatography coupled to tandem mass spectrometry, will be applied to capture the circulating sphingolipidome. Maximal cardiopulmonary exercise tests will be performed before and after the 8-week programme to assess patient fitness changes. Cholesterol, triglycerides, glycated haemoglobin, the homeostatic model assessment for insulin resistance, static retinal vessel analysis, flow-mediated dilatation, and strain analysis of the heart cavities will also be assessed pre- and post-intervention. This study shall inform whether and to what extent exercise can be used as an evidence-based treatment to lower circulating sphingolipid levels. The trial was registered on www.clinicaltrials.gov (NCT06024291) on August 28, 2023.

Alzheimer's disease manifests abnormal sphingolipid metabolism.

Alzheimer's disease (AD) is associated with disturbed metabolism, prompting investigations into specific metabolic pathways that may contribute to its pathogenesis and pathology. Sphingolipids have garnered attention due to their known physiological impact on various diseases. We conducted comprehensive profiling of sphingolipids to understand their possible role in AD. Sphingolipid levels were measured in AD brains, Cerad score B brains, and controls, as well as in induced pluripotent stem (iPS) cells (AD, PS, and control), using liquid chromatography mass spectrometry. AD brains exhibited higher levels of sphingosine (Sph), total ceramide 1-phosphate (Cer1P), and total ceramide (Cer) compared to control and Cerad-B brains. Deoxy-ceramide (Deoxy-Cer) was elevated in Cerad-B and AD brains compared to controls, with increased sphingomyelin (SM) levels exclusively in Cerad-B brains. Analysis of cell lysates revealed elevated dihydroceramide (dhSph), total Cer1P, and total SM in AD and PS cells versus controls. Multivariate analysis highlighted the relevance of Sph, Cer, Cer1P, and SM in AD pathology. Machine learning identified Sph, Cer, and Cer1P as key contributors to AD. Our findings suggest the potential importance of Sph, Cer1P, Cer, and SM in the context of AD pathology. This underscores the significance of sphingolipid metabolism in understanding and potentially targeting mechanisms underlying AD.

Sphingolipid profiling reveals differential functions of sphingolipid biosynthesis isozymes of Caenorhabditis elegans

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

Study protocol of the GRoningen early-PD Ambroxol treatment (GREAT) trial: a randomized, double-blind, placebo-controlled, single center trial with ambroxol in Parkinson patients with a GBA mutation.

To date, no disease modifying therapies are available for Parkinson's disease (PD). Since PD is the second most prevalent neurodegenerative disorder, there is a high demand for such therapies. Both environmental and genetic risk factors play an important role in the etiology and progression of PD. The most common genetic risk factor for PD is a mutation in the GBA1(GBA)-gene, encoding the lysosomal enzyme glucocerebrosidase (GCase). The mucolytic ambroxol is a repurposed drug, which has shown the property to upregulate GCase activity in-vitro and in-vivo. Ambroxol therefore has the potency to become a disease modifying therapy in PD, which was the reason to design this randomized controlled trial with ambroxol in PD patients. This trial is a single-center, double-blind, randomized, placebo-controlled study, including 80 PD patients with a GBA mutation, receiving either ambroxol 1800 mg/day or placebo for 48 weeks. The primary outcome measure is the Unified Parkinson's Disease Rating Scale motor subscore (part III) of the Movement Disorder Society (MDS-UPDRSIII) in the practically defined off-state at 60 weeks (after a 12-week washout period). Secondary outcomes include a 3,4-dihydroxy-6-18F-fluoro-I-phenylalanine ([18F]FDOPA) PET-scan of the brain, Magnetic Resonance Imaging (with resting state f-MRI and Diffusion Tensor Imaging), GCase activity, both intra- and extracellularly, sphingolipid profiles in plasma, Montreal Cognitive Assessment (MoCA), quality of life (QoL) measured by the Parkinson's Disease Questionnaire (PDQ-39) and the Non-Motor Symptom Scale (NMSS) questionnaire. Ambroxol up to 1200 mg/day has shown effects on human cerebrospinal fluid endpoints, which supports at least passage of the blood-brain-barrier. The dose titration in this trial up to 1800 mg/day will reveal if this dose level is safe and also effective in modifying the course of the disease. NCT05830396. Registration date: March 20, 2023.

Adipose tissue place of origin and obesity influence sphingolipid signaling pathway in the adipocytes differentiated from ADMSCs isolated from morbidly obese women

Adipose derived mesenchymal stem cells (ADMSCs) are a component of adipose tissue that in recent years has gained on importance. The progenitor cells serve as an essentially unlimited source of new adipocytes and therefore are considered to be an important determinant of the tissue’s physiology. In this paper we investigated mature adipocytes differentiated from ADMSCs obtained from subcutaneous/visceral fat of patients with different metabolic status (lean, obese without and with metabolic syndrome). We focused our interests on the sphingolipid signaling pathway, i.e.a signal transduction system indispensable for cells functioning, but also implicated in the development of medical conditions associated with obesity. We observed that the cells derived from visceral tissue had significantly greater levels of almost all the examined sphingolipids (especially Cer, dhCer, SM). Moreover, obesity and metabolic syndrome present in donor patients was associated with an increased level of sphingosine kinase (SPHK) and the product of its reaction sphingosine-1-phosphate (S1P). Moreover, the condition appeared to display a tissue specific pattern. Namely, the adipocytes of subcutaneous provenance had an increased activation of ceramide de novo synthesis pathway when the donors of ADMSCs had metabolic syndrome. The above translated into greater accumulation of ceramide in the cells. To our knowledge this is the first study that demonstrated altered sphingolipid profile in the mature adipocytes differentiated from ADMSCs with respect to the stem cells tissue of origin and the donor patient metabolic status.

Blood sphingolipid as a novel biomarker in patients with neuromyelitis optica spectrum disorder

BackgroundSphingolipids are signaling molecules and structural components of the axolemma and myelin sheath. Plasma sphingolipid levels may reflect disease status of neuromyelitis optica spectrum disorder (NMOSD). We aimed to examine plasma sphingolipids as disease severity biomarkers for NMOSD and compare their characteristics with those of serum neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP). MethodsWe measured plasma sphingolipids, sNfL, and sGFAP levels in NMOSD cases with anti-aquaporin-4-antibody. An unbiased approach, partial least square discriminant analysis (PLS-DA), was utilized to determine whether sphingolipid profiles differ according to the disease state of NMOSD (presence, moderate-to-severe disability [Expanded Disease Severity Scale, (EDSS) > 3.0], and relapses). ResultsWe investigated 81 patients and 10 controls. PLS-DA models utilizing sphingolipids successfully differentiated patients with EDSS > 3.0, but failed to identify the presence of disease and relapses. Ceramide-C14—a significant contributor to differentiating EDSS > 3.0—positively correlated with EDSS, while its levels were independent of age and the presence of relapses. This characteristic was unique from those of sNfL and sGFAP, which were affected by age and relapses as well as EDSS. ConclusionPlasma sphingolipids may be useful NMOSD biomarkers for disability with distinct characteristics compared to sNfL and sGFAP.

Plasma Ceramides and Other Sphingolipids in Relation to Incident Prediabetes in a Longitudinal Biracial Cohort.

Sphingolipids are linked to the pathogenesis of type 2 diabetes. To test the hypothesis that plasma sphingolipid profiles predict incident prediabetes. A case-control study nested in the Pathobiology of Prediabetes in a Biracial Cohort study, a 5-year follow-up study. Academic health center. Normoglycemic adults enrolled in the Pathobiology of Prediabetes in a Biracial Cohort study. Assessments included oral glucose tolerance test, insulin sensitivity, and insulin secretion. Participants with incident prediabetes were matched in age, sex, and ethnicity with nonprogressors. We assayed 58 sphingolipid species (ceramides, monohexosyl ceramides, sphingomyelins, and sphingosine) using liquid chromatography/tandem mass spectrometry in baseline plasma levels from participants and determined association with prediabetes risk. The primary outcome was progression from normoglycemia to prediabetes, defined as impaired fasting glucose or impaired glucose tolerance. The mean age of participants (N = 140; 50% Black, 50% female) was 48.1 ± 8.69 years, body mass index 30.1 ± 5.78 kg/m2, fasting plasma glucose 92.7 ± 5.84 mg/dL, and 2-hour plasma glucose 121 ± 23.3 mg/dL. Of the 58 sphingolipid species assayed, higher ratios of sphingomyelin C26:0/C26:1 (OR, 2.73 [95% CI, 1.172-4.408], P = .015) and ceramide C18:0/C18:1 (OR, 1.236 [95% CI, 1.042-1.466], P = .015) in baseline plasma specimens were significantly associated with progression to prediabetes during the 5-year follow-up period, after adjustments for age, race, sex, body mass index, fasting plasma glucose, 2-hour plasma glucose, insulin sensitivity, and insulin secretion. We conclude that the saturated-to-monounsaturated ratios of long-chain ceramide C18:0/C18:1 and very-long-chain sphingomyelin C26:0/C26:1 are potential biomarkers of prediabetes risk among individuals with parental history of type 2 diabetes.

Development of an LC-MS/MS Method to Measure Sphingolipids in CSF from Patients with Multiple Sclerosis.

Multiple sclerosis is an inflammatory and degenerative disease characterized by different clinical courses including relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). A hallmark of patients with multiple sclerosis (pwMS) includes a putative autoimmune response, which results in demyelination and neuroaxonal damage in the central nervous system. Sphingolipids in cerebrospinal fluid (CSF) have been proposed as potential biomarkers reflective of disease activity in pwMS. Hence, sensitive methods to accurately quantify sphingolipids in CSF are needed. In this study, we report the development of a sensitive high-throughput multiplexed liquid chromatography coupled to a tandem mass spectrometry method to perform quantitation on 14 species of sphingolipids in human CSF. We applied this method to measure CSF sphingolipids in healthy controls (n = 10), PPMS (n = 27), and RMS (n = 17) patients before and after ocrelizumab treatment. The median CSF levels of the 14 sphingolipids measured herein was higher in PPMS (17.2 ng/mL) and RMS (17.6 ng/mL) when compared with the healthy controls (13.8 ng/mL). Levels of sphingolipids were decreased by 8.6% at week 52 after treatment with ocrelizumab in RMS patients but not in PPMS patients. Specifically, C16 glucosylceramide (-26%; P = 0.004) and C18 ceramides (-13%; P = 0.042) decreased from baseline in RMS patients. Additionally, in PPMS patients C16 glucosylceramide levels correlated with CSF neurofilament heavy levels at baseline (Rho =0.532; P = 0.004) and after treatment (Rho =0.424; P = 0.028). Collectively, these results indicate that CSF sphingolipid levels are altered in pwMS and treatment with ocrelizumab results in significant shifts in the sphingolipid profile that may reflect a reduction in disease activity supporting further investigation into sphingolipids as tools to monitor disease state. SIGNIFICANCE STATEMENT: This study describes the development of a new method to measure 14 sphingolipid species in CSF. These results demonstrate that sphingolipids levels are elevated in CSF from pwMS compared to healthy controls. Distinct sphingolipid signatures were observed between patients with different clinical disease courses, and these lipid signatures changed after treatment with ocrelizumab, especially in RMS patients. This method enables further investigation into the role of sphingolipids as candidate biomarkers in pwMS and other central nervous system disorders.