Lack of ceramide synthase 2 suppresses the development of experimental autoimmune encephalomyelitis by impairing the migratory capacity of neutrophils

Ceramide synthases (CerS) synthesise ceramides of defined acyl chain lengths, which are thought to mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed a significant elevation of CerS2 and its products, C24-ceramides, in CD11b⁺ cells (monocytes and neutrophils) isolated from blood. This result correlates with the clinical finding that CerS2 mRNA expression and C24-ceramide levels were significantly increased by 2.2- and 1.5-fold, respectively, in white blood cells of MS patients. The increased CerS2 mRNA/C24-ceramide expression in neutrophils/monocytes seems to mediate pro-inflammatory effects, since a specific genetic deletion of CerS2 in blood cells or a total genetic deletion of CerS2 significantly delayed the onset of clinical symptoms, due to a reduced infiltration of immune cells, in particular neutrophils, into the central nervous system. CXCR2 chemokine receptors, expressed on neutrophils, promote the migration of neutrophils into the central nervous system, which is a prerequisite for the recruitment of further immune cells and the inflammatory process that leads to the development of MS. Interestingly, neutrophils isolated from CerS2 null EAE mice, as opposed to WT EAE mice, were characterised by significantly lower CXCR2 receptor mRNA expression resulting in their reduced migratory capacity towards CXCL2. Most importantly, G-CSF-induced CXCR2 expression was significantly reduced in CerS2 null neutrophils and their migratory capacity was significantly impaired. In conclusion, our data strongly indicate that G-CSF-induced CXCR2 expression is regulated in a CerS2-dependent manner and that CerS2 thereby promotes the migration of neutrophils, thus, contributing to inflammation and the development of EAE and MS.     

丝裂霉素C与C2-神经酰胺联合应用治疗膀胱癌

目的 观察丝裂霉素C（MMC）与C2-神经酰胺（C2-cer）联合应用对人膀胱癌细胞的作用效果，并探讨其机制。方法 不同浓度MMC与C2-cer单独及联合作用于人膀胱癌BIU-87细胞后，应用噻唑蓝（MTT）比色法检测细胞生长抑制率，计算合用指数（CI），流式细胞仪（FCM）检测BIU-87细胞凋亡率，吖啶橙（AO）荧光染色观察凋亡形态学变化，Western blot检测细胞色素C在细胞内分布变化，并检测Caspase-3活性改变。结果 单独应用时MMC与C2-cer的中效浓度分别是159和28μmol／L，联合用药时下降为55和11μmol／L，CI=0．74。MMC与C2-cer单独及联合应用均可导致BIU-87细胞出现凋亡的形态学变化。两种药物联合应用时的凋亡率高于各自单用（P＜0．05）。线粒体细胞色素C含量在MMC与C2-cer单独及联合应用时均较对照组减少，联合用药时减少最为明显，细胞质内细胞色素C含量在联合用药时增加也最为明显（P＜0．05）。Caspase-3活性在MMC与C2-cer单独及联合应用时均较对照组升高，联合用药时升高最为明显（P＜0．05）。结论 MMC与C2-cer联合应用可以通过共同诱导细胞凋亡，协同抑制膀胱癌细胞生长。线粒体细胞色素C释放和Caspase-3活性变化可能发挥重要作用。     

Metabolism of Very Long-Chain Fatty Acids: Genes and Pathophysiology

Fatty acids (FAs) are highly diverse in terms of carbon (C) chain-length and number of double bonds. FAs with C>20 are called very long-chain fatty acids (VLCFAs). VLCFAs are found not only as constituents of cellular lipids such as sphingolipids and glycerophospholipids but also as precursors of lipid mediators. Our understanding on the function of VLCFAs is growing in parallel with the identification of enzymes involved in VLCFA synthesis or degradation. A variety of inherited diseases, such as ichthyosis, macular degeneration, myopathy, mental retardation, and demyelination, are caused by mutations in the genes encoding VLCFA metabolizing enzymes. In this review, we describe mammalian VLCFAs by highlighting their tissue distribution and metabolic pathways, and we discuss responsible genes and enzymes with reference to their roles in pathophysiology.     

Sphingomyelin changes in rat cerebral cortex during focal ischemia

Abstract

To better define the sphingolipid metabolism during focal brain ischemia, levels of ceramide, sphingomyelin, cerebroside and gangliosides were determined in rat cerebral cortex during focal ischemia produced by occlusion of the middle cerebral artery. Sphingomyelin began to decrease at 2 hours of ischemia and continued to decrease for 96 hours. In contrast, ceramide increased at 6 hours and increased to 4.2-fold at 96 hours after ischemia, and the fatty acid composition of ceramide was solely nonhydroxylated fatty acid similar to sphingomyelin. Hydroxylated fatty acid-linked cerebroside decreased at 6 hours of ischemia, whereas any significant decrease of nonhydroxylated fatty acid-linked cerebroside didn't occur for 96 hours of ischemia. There were no measurable changes in the levels of gangliosides. These results suggested that ceramide was produced in the cerebral cortex by the breakdown of sphingomyelin during early ischemia. [Neurol Res 1996; 18: 337-341]     

Hypomyelinating spastic dyskinesia and ichthyosis caused by a homozygous splice site mutation leading to exon skipping in ELOVL1

IntroductionNext generation sequencing technologies allow detection of very rare pathogenic gene variants and uncover cerebral palsy. Herein, we describe two siblings with cerebral palsy due to ELOVL1 splice site mutation in autosomal recessive manner. ELOVL1 catalyzes fatty acid elongation to produce very long-chain fatty acids (VLCFAs; ≥C21), most of which are components of sphingolipids such as ceramides and sphingomyelins. Ichthyotic keratoderma, spasticity, hypomyelination, and dysmorphic facies (MIM: 618527) stem from ELOVL1 gene deficiency in human.MethodsWe have studied a consanguineous family with whole exome sequencing (WES) and performed in depth analysis of cryptic splicing on the molecular level using RNA. Comprehensive analysis of ceramides in the skin stratum corneum of patients using liquid chromatography-tandem mass spectrometry (LC–MS/MS). ELOVL1 protein structure was computationally modelled.ResultsThe novel c.376-2A > G (ENST00000372458.8) homozygous variant in the affected siblings causes exon skipping. Comprehensive analysis of ceramides in the skin stratum corneum of patients using LC–MS/MS demonstrated significant shortening of fatty acid moieties and severe reduction in the levels of acylceramides.DiscussionIt has recently been shown that disease associated variants of ELOVL1 segregate in an autosomal dominant manner. However, our study for the first time demonstrates an alternative autosomal recessive inheritance model for ELOVL1. In conclusion, we suggest that in ultra-rare diseases, being able to identify the inheritance patterns of the disease-associated gene or genes can be an important guide to identifying the molecular mechanism of genetic cerebral palsy.     

Autocrine motility-stimulatory pathways of oral premalignant lesion cells

Patients with premalignant oral lesions have varying levels of risk of developing oral squamous cell carcinoma (OSCC), whose aggressiveness requires increased motility. Not known is if and how premalignant oral lesion cells acquire the increased motility characteristic of OSCC. This was addressed by immunohistochemical analysis of banked premalignant lesion tissues and by functional analyses using cultures established from premalignant oral lesions and OSCC. These studies showed premalignant oral lesion cells and OSCC to be more motile than normal keratinocytes. Concomitantly, levels of ceramide were reduced. The activity of the protein phosphatase PP-2A, which restricts motility and which can be activated by ceramide, was also diminished. This was due to IL-10 released from premalignant lesion cells. Treatment with a membrane-permeable ceramide restored PP-2A activity and blocked migration. These studies show an autocrine motility-stimulatory pathway that is mediated in premalignant lesion cells by IL-10 through its reduction of ceramide levels and inhibition of PP-2A activity.     

Differential effects of ceramide species on exocytosis in rat PC12 cells

Increases in several ceramide species have been shown by non-targeted lipid profiling (lipidomics) of the rat hippocampus after kainate lesions (Guan et al. FASEB J 20:1152–1161, 2006). This study was carried out to examine possible effects of ceramide species on exocytosis. Significant increase in membrane capacitance in voltage-clamped rat pheochromocytoma (PC12) cells, an indication of exocytosis, was detected immediately after external application of C2, C6, and C18 ceramide. In contrast, no increase in capacitance was found after addition of C16 and C20 ceramide, or DMSO vehicle. The effect of ceramide on exocytosis was dependent on the integrity of lipid rafts. Treatment of cells with the cholesterol binding agent/disruptor of lipid rafts, methyl β cyclodextrin, prior to addition of C18 ceramide suppressed the increase in capacitance induced by this lipid species. The ability of C2, C6 and C18 ceramide to trigger exocytosis was confirmed using total internal reflection fluorescence microscopy (TIRFM) experiments. External application of these species caused an exponential decrease in the number of subplasmalemmal neuropeptide Y (NPY)-enhanced green fluorescence protein (EGFP) labeled vesicles, indicating exocytosis. Interestingly, C18 is also the ceramide species that showed the greatest increase in the rat hippocampus after kainate excitotoxicity. It is postulated that C18 ceramide might facilitate exocytosis of glutamate from damaged neurons, thus propagating neuronal injury.     

度普利尤单抗对成人中重度特应性皮炎 皮肤屏障功能的影响

目的 探讨度普利尤单抗对中重度特应性皮炎（atopic dermatitis,AD）患者皮肤屏障功能的影响。方法选取2020年9月至2021年10月于锦州医科大学附属第一医院就诊的20例成人中重度AD患者纳入研究组,同期于该院体检的20名健康志愿者纳入对照组。研究组患者给予度普利尤单抗治疗,分别于治疗前和治疗8周、12周、16周后检测AD患者前臂掌侧皮损部位皮肤含水量与经皮水分丢失（transepidermal water loss,TEWL）,及距离此部位5cm的非皮损部位角质层神经酰胺（ceramide,Cer）含量;相同方法检测对照组研究对象的皮肤含水量、TEWL和皮肤角质层Cer含量。结果 研究组共18例患者完成研究。研究组患者治疗后,EASI评分逐渐下降,治疗8周、12周、16周后EASI评分均显著低于治疗前（P<0.05）。研究组患者治疗前的Cer含量、皮肤含水量均显著低于对照组（P<0.05）,TEWL显著高于对照组（P<0.05）;治疗16周后,两组研究对象的TEWL比较差异无统计学意义（P>0.05）。治疗后,研究组患者的皮肤角质层Cer含量、皮肤含水量逐渐升高,治疗12周、16周后上述指标均显著高于治疗前（P<0.05）。治疗后,研究组患者的TEWL逐渐降低,治疗8周、12周、16周后TEWL均显著低于治疗前（P<0.05）。皮肤角质层Cer含量、皮肤含水量与EASI均呈负相关（P<0.001）,TEWL与EASI呈正相关（P<0.001）。结论 度普利尤单抗可通过减少TEWL、增加皮肤含水量和角质层Cer含量,改善皮肤屏障功能。     

Ceramides are associated with inflammatory processes in human mediastinal adipose tissue

Background and AimCeramides are poorly characterized in human adipose tissue. The aim of this study was to investigate concentrations of different ceramide species in human subcutaneous and visceral adipose tissue depots and to determine associations between ceramides and global gene expression profiles.Methods and ResultsConcentrations of six ceramide species were determined in plasma and in subcutaneous and mediastinal adipose tissue from 10 overweight subjects (BMI 29.4 ± 4.9 kg/m²). In the adipose tissue biopsies gene expression arrays were performed and relationships between ceramides and gene expression analyzed. Immunostaining of the two adipose tissue depots was performed in an independent group of 10 patients. Mediastinal adipose tissue contained significantly higher concentrations (p < 0.05) of all six ceramide species than the subcutaneous depot. Of the six ceramides in plasma, concentrations of only two (Cer d18:1/18:0 and Cer d18:1/22:0) correlated significantly (p < 0.05) with the corresponding species in mediastinal adipose tissue, but there were no significant correlations between ceramides in plasma and subcutaneous adipose tissue. Multivariate analysis identified significant correlations between the total ceramide concentration and global gene expression within mediastinal, but not subcutaneous adipose tissue, according to cross-validation. Gene ontology analysis of genes related to ceramides in the mediastinal depot revealed that genes positively correlated with ceramides were associated mainly with immune and inflammatory categories, while genes negatively correlated with ceramides were associated mainly with lipid and carbohydrate metabolism.ConclusionsCeramides in human mediastinal adipose tissue may be involved in inflammation and lipid and carbohydrate metabolism.