Dysregulation Of Sphingolipid Metabolism Research Articles

EPEN-14. A Phase 1, Safety and Dose Escalation Study of BXQ-350 in Patients with Advanced Solid Malignancies Demonstrates that BXQ-350 is Well Tolerated and Shows Signs of Potential Clinical Activity in Ependymoma Patients

Abstract The significance of a dysregulated sphingolipid metabolism in cancer, including brain tumors, has been demonstrated and several enzymes involved in sphingolipid metabolism are being investigated as novel therapeutic targets for adult and pediatric brain cancers. Saposin C (Sphingolipid Activator PrO[S]etIN) is a human protein encoded by the Psap gene and is an allosteric activator of several enzymes involved in sphingolipid/ceramide metabolism. BXQ-350 is a nanovesicle of Saposin C that has broad anticancer activity, selectively inducing apoptosis of cancer cells by lowering Sphingosine-1-Phosphate and increasing ceramides concentrations and inducing an anti-tumoral immune response. BXQ-350 was investigated in a Phase 1 dose-escalation safety study in cancer patients with advanced solid malignancies including brain tumors (NCT02859857). The primary objective of this single agent study was to describe the safety profile and to determine the maximum tolerated dose, or the biological effective dose, of BXQ-350 administered intravenously at escalating doses from 0.7 mg/kg up to 2.4 mg/kg. Multiple secondary parameters were included to characterize BXQ-350’s pharmacokinetic parameters, efficacy profile and potential biomarkers. This trial was performed at four US sites. Results indicate that BXQ-350 was safe and well-tolerated as no DLT was observed and an MTD was not reached. RANO or RECIST 1.1 criteria were used to evaluate tumor response. Analysis of plasma samples suggests that BXQ-350 modulates sphingolipid metabolism and impacts the immune system positively. Furthermore, potential signs of single agent activity were observed across tumor types, including in two ependymomas for which results will be presented.

Pharmacological sphingosine-1 phosphate receptor 1 targeting in cigarette smoke-induced emphysema in mice.

Primarily caused by chronic cigarette smoking (CS), emphysema is characterized by loss of alveolar cells comprising lung units involved in gas exchange and inflammation that culminate in airspace enlargement. Dysregulation of sphingolipid metabolism with increases of ceramide relative to sphingosine-1 phosphate (S1P) signaling has been shown to cause lung cell apoptosis and is emerging as a potential therapeutic target in emphysema. We sought to determine the impact of augmenting S1P signaling via S1P receptor 1 (S1P1) in a mouse model of CS-induced emphysema. DBA2 mice were exposed to CS for 4 or 6 mo and treated with pharmacological agonists of S1P1: phosphonated FTY720 (FTY720-1S and 2S analogs; 0.01-1.0 mg/kg) or GSK183303A (10 mg/kg). Pharmacological S1P1 agonists ameliorated CS-induced lung parenchymal apoptosis and airspace enlargement as well as loss of body weight. S1P1 agonists had modest inhibitory effects on CS-induced airspace inflammation and lung functional changes measured by Flexivent, improving lung tissue resistance. S1P1 abundance was reduced in chronic CS-conditions and remained decreased after CS-cessation or treatment with FTY720-1S. These results support an important role for S1P-S1P1 axis in maintaining the structural integrity of alveoli during chronic CS exposure and suggest that increasing both S1P1 signaling and abundance may be beneficial to counteract the effects of chronic CS exposure.

Grade-dependent changes in sphingolipid metabolism in clear cell renal cell carcinoma.

There is a host of evidence for the role of bioactive sphingolipids in cancer biology, and dysregulated sphingolipid metabolism was observed in many malignant tumors. The aim of the present study was to provide more detailed data on sphingolipid metabolism in different stages of clear cell renal cell carcinoma (ccRCC). Samples of the tumor and noncancerous fragments of the same kidney were collected from patients who underwent a radical nephrectomy. The subjects were stratified according to the degree of malignancy of the tumor (n = 14 for G2, 12 for G3, and 9 for G4). The content of bioactive sphingolipids/glycosphingolipids was measured with an HPLC and HPTLC method, and the mRNA and protein expression of sphingolipid transporters and metabolizing enzymes was evaluated using real-time polymerase chain reaction (PCR) and Western blot, respectively. Compared to healthy kidney tissue, ccRCC was characterized by accumulation of sphingosine, sphingosine-1-phosphate (S1P), ceramide, dihydrosphingosine, and dihydroceramide. However, in the case of the latter two, the accumulation was limited to higher malignancy grades. In addition, compared to the healthy tissue, the content of gangliosides in the tumor was increased at the expense of globosides. We also found profound grade-dependent changes in the mRNA level of S1P-metabolizing enzymes, and spinster homolog 2. In general, their expression was much higher in G2 tumors compared to higher malignancy grades. We conclude that ccRCC is characterized by profound and multilevel alterations in sphingolipid metabolism, which to a large extent are grade-dependent. We hypothesize that dysregulation of sphingolipid metabolism contributes to the progression of ccRCC.

Dysregulation in Sphingolipid Signaling Pathways is Associated With Symptoms and Functional Connectivity of Pain Processing Brain Regions in Provoked Vestibulodynia

Provoked vestibulodynia (PVD) is a chronic pain disorder characterized by local hypersensitivity and severe pain with pressure localized to the vulvar vestibule. Despite decades of study, the lack of identified biomarkers has slowed the development of effective therapies. The primary aim of this study was to use metabolomics to identify novel biochemical mechanisms in vagina and blood underlying brain biomarkers and symptoms in PVD, thereby closing this knowledge gap. Using a cross-sectional case-control observational study design, untargeted and unbiased metabolomic profiling of vaginal fluid and plasma was performed in women with PVD compared to healthy controls. In women with PVD, we also obtained assessments of vulvar pain, vestibular and vaginal muscle tenderness, and 24-hour symptom intensity alongside resting-state brain functional connectivity of brain regions involved in pain processing and modulation. Compared to healthy controls, women with PVD demonstrated differences primarily in vaginal (but not plasma) concentrations of metabolites of the sphingolipid signaling pathways, suggesting localized effects in vagina and vulvar vestibule rather than systemic effects. Our findings reveal that dysregulation of sphingolipid metabolism in PVD is associated with increased vulvar pain and muscle tenderness, sexual dysfunction, and decreased functional connectivity strength in pain processing/modulatory brain regions. This data collectively suggests that alterations in sphingolipid signaling pathways are likely an important molecular biomarker in PVD that could lead to new targets for therapeutic intervention. PerspectiveThis manuscript presents the results of a robust, unbiased molecular assessment of plasma and vaginal fluid samples in women with provoked vestibulodynia compared to healthy controls. The findings suggest that alterations in sphingolipid signaling pathways are associated with symptoms and brain biomarkers and may be an important molecular marker that could provide new targets for therapeutic intervention.

Sortilin evokes endothelial dysfunction and arterial hypertension through the dysregulation of sphingolipid metabolism and oxidative stress

Abstract Background Sortilin, a member of vacuolar protein sorting domain family Vps10, has been positively correlated with vascular and metabolic disorders in humans. Previous study has shown that, in response to Fas receptor stimulation, sortilin together with acid sphingomyelinase (ASMase) promote the clustering of lipid rafts and subsequent activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in coronary endothelial cells. However, whether sortilin plays a role in endothelial cells function is currently unknown. Purpose To assess whether sortilin per se was able to influence vascular function, thereby contributing to the pathogenesis of cardiovascular diseases. Methods Pressure myography was used to study vascular reactivity of mesenteric arteries. To investigate the involvement of acid sphingomyelinase (ASM), we performed gene silencing approach and fluorometric activity assay. NADPH oxidase lucigenin assay was used to evaluate oxidative stress in endothelial cells and resistance vessels. The effects of circulating sortilin on cardiovascular system was evaluated by systemic delivery of recombinant sortilin protein to wild-type (WT), sphingosine-1-phosphate receptor 3 (S1P3) and NADPH oxidase 2 (gp91phox/NOX2) deficient mice. Systolic arterial blood pressure (SBP) was noninvasively registered in conscious mice by tail-cuff blood monitoring. Finally, to explore the translational relevance of sortilin, we measured sortilin and NOX2 soluble derived peptide levels using ELISA and quantified sphingosine-1-phosphate (S1P) by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in plasma of hypertensive patients. Results Here we demonstrated that sortilin evoked endothelial dysfunction in mesenteric arteries due to increased NADPH oxidase-derived oxidative stress. Knockdown of ASM successfully prevented impairment of endothelial function. Using the inhibitor of sphingosine kinase type 1 (SphK1), sortilin failed to evoke endothelial impairment as well as NADPH oxidase activation. In endothelial cells, sortilin induced S1P-dependent activation of Rac1/NOX2 signaling axis, which was prevented by TY-52156, an antagonist of lysosphingolipid receptor S1P3. In vivo sortilin administration induced arterial hypertension in WT mice. In contrast, genetic deletion of S1P3 and gp91phox/NOX2 resulted in preservation of endothelial function and SBP unchanged levels after 14 days of systemic sortilin administration. Finally, to translate these research findings into a clinical setting, we found that hypertensive patients have higher plasma levels of sortilin, ASMase, S1P and soluble NOX2 derived peptide than normotensive subjects. Conclusions These results demonstrate the pathologic role of sortilin in the modulation of endothelial function and arterial blood pressure, suggesting that sortilin and its mediators might represent novel therapeutic targets in vascular diseases and hypertension. Funding Acknowledgement Type of funding source: None

Intestinal Acid Sphingomyelinase Protects From Severe Pathogen-Driven Colitis.

Inflammatory diseases of the gastrointestinal tract are emerging as a global problem with increased evidence and prevalence in numerous countries. A dysregulated sphingolipid metabolism occurs in patients with ulcerative colitis and is discussed to contribute to its pathogenesis. In the present study, we determined the impact of acid sphingomyelinase (Asm), which catalyzes the hydrolysis of sphingomyelin to ceramide, on the course of Citrobacter (C.) rodentium-driven colitis. C. rodentium is an enteric pathogen and induces colonic inflammation very similar to the pathology in patients with ulcerative colitis. We found that mice with Asm deficiency or Asm inhibition were strongly susceptible to C. rodentium infection. These mice showed increased levels of C. rodentium in the feces and were prone to bacterial spreading to the systemic organs. In addition, mice lacking Asm activity showed an uncontrolled inflammatory Th1 and Th17 response, which was accompanied by a stronger colonic pathology compared to infected wild type mice. These findings identified Asm as an essential regulator of mucosal immunity to the enteric pathogen C. rodentium.

Reduced Serum Sphingolipids Constitute a Molecular Signature of Malnutrition in Hospitalized Patients With Decompensated Cirrhosis.

INTRODUCTION:Malnutrition is a leading cause of morbidity and mortality in cirrhosis. Although multiple noninvasive measures of nutritional status have been studied, no consensus exists for early identification of malnutrition in cirrhosis. Serum metabolomics offers a novel approach for identifying biomarkers in multiple disease states. To characterize alterations in metabolic pathways associated with malnutrition in hospitalized cirrhotic patients and to identify biomarkers for disease prognosis.METHODS:In this cross-sectional, observational cohort study, 51 hospitalized cirrhotic patients were classified as malnourished (42.3%) or nourished (57.7%) based on low mid-arm muscle circumference and dominant handgrip strength. Anthropometric measurements and computed tomography body composition analysis were performed. Serum was collected after overnight fasting for unbiased metabolomics analysis.RESULTS:Malnourished cirrhotic patients exhibited mild reductions in skeletal muscle index, with more marked reductions in visceral fat index. Seventy-one biochemicals were significantly altered in malnourished subjects. The serum metabolite profile was significantly different between nourished and malnourished cirrhotic patients. Pathway analysis demonstrated that only sphingolipid metabolic pathways were significantly enriched in altered metabolites. Hierarchical clustering revealed that sphingolipid metabolites clustered into nourished and malnourished cohorts. Spearman analysis demonstrated multiple statistically significant correlations between sphingolipid species and Model for End-Stage Liver Disease-Sodium. Using logistic regression, we identified 8 sphingolipids that were significantly associated with malnutrition after controlling for Model for End-Stage Liver Disease-Sodium, age, and gender.CONCLUSIONS:Malnutrition in hospitalized cirrhotic patients is characterized by reductions in multiple sphingolipid species. Dysregulated sphingolipid metabolism may be involved in the pathophysiology of malnutrition in cirrhosis and potentially serve as a biomarker of nutritional status in this population.

Identifying therapeutic targets by combining transcriptional data with ordinal clinical measurements

The immense and growing repositories of transcriptional data may contain critical insights for developing new therapies. Current approaches to mining these data largely rely on binary classifications of disease vs. control, and are not able to incorporate measures of disease severity. We report an analytical approach to integrate ordinal clinical information with transcriptomics. We apply this method to public data for a large cohort of Huntington’s disease patients and controls, identifying and prioritizing phenotype-associated genes. We verify the role of a high-ranked gene in dysregulation of sphingolipid metabolism in the disease and demonstrate that inhibiting the enzyme, sphingosine-1-phosphate lyase 1 (SPL), has neuroprotective effects in Huntington’s disease models. Finally, we show that one consequence of inhibiting SPL is intracellular inhibition of histone deacetylases, thus linking our observations in sphingolipid metabolism to a well-characterized Huntington’s disease pathway. Our approach is easily applied to any data with ordinal clinical measurements, and may deepen our understanding of disease processes.

Sphingolipid accumulation causes mitochondrial dysregulation and cell death.

Sphingolipids are structural components of cell membranes that have signaling roles to regulate many activities, including mitochondrial function and cell death. Sphingolipid metabolism is integrated with numerous metabolic networks, and dysregulated sphingolipid metabolism is associated with disease. Here, we describe a monogenic yeast model for sphingolipid accumulation. A csg2Δ mutant cannot readily metabolize and accumulates the complex sphingolipid inositol phosphorylceramide (IPC). In these cells, aberrant activation of Ras GTPase is IPC-dependent, and accompanied by increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial mass. Survival or death of csg2Δ cells depends on nutritional status. Abnormal Ras activation in csg2Δ cells is associated with impaired Snf1/AMPK protein kinase, a key regulator of energy homeostasis. csg2Δ cells are rescued from ROS production and death by overexpression of mitochondrial catalase Cta1, abrogation of Ras hyperactivity or genetic activation of Snf1/AMPK. These results suggest that sphingolipid dysregulation compromises metabolic integrity via Ras and Snf1/AMPK pathways.

Sphingolipids Are Dual Specific Drug Targets for the Management of Pulmonary Infections: Perspective.

Sphingolipids are the major constituent of the mucus secreted by the cells of epithelial linings of lungs where they maintain the barrier functions and prevent microbial invasion. Sphingolipids are interconvertible, and their primary and secondary metabolites have both structural and functional roles. Out of several sphingolipid metabolites, sphingosine-1 phosphate (S1P) and ceramide are central molecules and decisive for sphingolipid signaling. These are produced by enzymatic activity of sphingosine kinase-1 (SK-1) upon the challenge with either biological or physiological stresses. S1P and ceramide rheostat are important for the progression of various pathologies, which are manifested by inflammatory cascade. S1P is a well-established secondary messenger and associated with various neuronal, metabolic, and inflammatory diseases other than respiratory infections such as Chlamydia pneumoniae, Streptococcus pneumoniae, and Mycobacterium tuberculosis. These pathogens are known to exploit sphingolipid metabolism for their opportunistic survival. Decreased sphingosine kinase activity/S1P content in the lung and peripheral blood of tuberculosis patients clearly indicated a dysregulation of sphingolipid metabolism during infection and suggest that sphingolipid metabolism is important for management of infection by the host. Our previous study has demonstrated that gain of SK-1 activity is important for the maturation of phagolysosomal compartment, innate activation of macrophages, and subsequent control of mycobacterial replication/growth in macrophages. Furthermore, S1P-mediated amelioration of lung pathology and disease severity in TB patients is believed to be mediated by the selective activation or rearrangement of various S1P receptors (S1PR) particularly S1PR2, which has been effective in controlling respiratory fungal pathogens. Therefore, such specificity of S1P–S1PR would be paramount for triggering inflammatory events, subsequent activation, and fostering bactericidal potential in macrophages for the control of TB. In this review, we have discussed and emphasized that sphingolipids may represent effective novel, yet dual specific drug targets for controlling pulmonary infections.

Altered Levels of Serum Ceramide, Sphingosine and Sphingomyelin Are Associated with Colorectal Cancer: A Retrospective Pilot Study.

Because patients with cancer of apparently equivalent stage often have different outcomes, it is necessary to gather additional information to complement cancer staging. Dysregulated sphingolipid metabolism contributes to carcinogenesis. In this retrospective pilot study, we tested the hypothesis that changes in serum levels of sphingolipids are associated with stage IV colorectal cancer (CRC). We used commercially available serum samples from healthy males and patients with CRC (adenocarcinoma of the large intestine, stage IV with metastases). Blood samples were obtained immediately prior to anesthesia/surgery. We measured sphingolipid levels in sera using mass spectrometry. In serum of patients with CRC, the levels of C16-, C18-, C18:1-, and C24:1-ceramide, as well as those of sphingosine, were significantly higher than those of controls. In contrast, the levels of C24-sphingomyelin were significantly lower than those of controls. A global test of association showed that ceramides and sphingomyelins but not hexosylceramides were significantly associated with stage IV CRC. Sphingolipids have a potential of serving as novel, non-invasive, inexpensive, and effective blood-based biomarkers to complement CRC staging for better prognosis and more personalized medicine.

Aberrant TGFβ Signalling Contributes to Dysregulation of Sphingolipid Metabolism in Intrauterine Growth Restriction.

Sphingolipids function as key bioactive mediators that regulate cell fate events in a variety of systems. Disruptions in sphingolipid metabolism characterize several human pathologies. In the present study we examined sphingolipid metabolism in intrauterine growth restriction (IUGR), a severe disorder complicating 4-7% of pregnancies at increased risk of perinatal morbidity and mortality, which is characterized by placental dysfunction and augmented trophoblast cell death rates. Placentae from early severe IUGR with documented abnormal umbilical artery Doppler defined as absence or reverse of end diastolic velocity and a birth weight below the fifth percentile for gestational age were collected (n = 58). Placental tissues obtained from healthy, age-matched preterm and term deliveries (n = 46; TC, n=28) were included as controls. Sphingolipid analysis by tandem mass spectrometry revealed elevated sphingosine and decreased ceramide levels in placentae from pregnancies complicated by IUGR relative to age-matched controls. Sphingosine accumulation was due to accelerated ceramide breakdown via increased acid ceramidase (ASAH1) expression/activity caused by augmented TGFβ signalling via the ALK5/SMAD2 pathway. In addition, a marked reduction in sphingosine kinase 1 (SPHK1) expression/activity due to impaired TGFβ signalling via ALK1/SMAD1 contributed to the sphingosine buildup in IUGR. Of clinical significance, ALK/SMAD signalling pathways were differentially altered in IUGR placentae. Altered TGFβ signalling in IUGR placentae causes dysregulation of sphingolipid metabolism, which may contribute to the increased trophoblast cell death typical of this pathology.

Sphingosine kinase 2 and sphingosine-1-phosphate promotes mitochondrial function in dopaminergic neurons of mouse model of Parkinson’s disease and in MPP+-treated MN9D cells in vitro

Dysregulation of sphingolipid metabolism has been shown to trigger the pathophysiology of many neurodegenerative disorders. The present study focuses on the role of one of the two sphingosine kinases, Sphk2 and its metabolite sphingosine-1-phosphate (S1P) signaling in Parkinson’s disease (PD). Our study indicated a marked down regulation of Sphk2 expression in the substantia nigra region of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and in the cellular PD model. Localization studies indicated that Sphk2 was predominantly present in mitochondria, proposing for its potential role in mitochondrial functions. Since mitochondrial dysfunction has been described to be the major pathological event in PD, the present study focused on the role of Sphk2/S1P signaling in promoting mitochondrial functions in the MPTP-induced mouse model of PD and in 1-methyl-4 phenylpyridinium (MPP+)-treated MN9D cells. Our study demonstrated that inhibition of Sphk2 decreased the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and its downstream targets nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM) which are the key genes regulating mitochondrial function. In addition, there was also a significant reduction in the total cellular adenosine triphosphate (ATP) and superoxide dismutase 2 (SOD 2) with an associated increase in levels of reactive oxygen species (ROS) in the absence of Sphk2. Interestingly, it was found that treating the cells with exogenous S1P along with MPP+ exerted a neuroprotective effect by activation of p-CREB, PGC-1α and NRF-1 in the MN9D cells. Moreover, the level of ATP was unaffected in the MPP+-treated cells in the presence of S1P. It was also observed that levels of ROS were significantly decreased in the MPP+-treated cells in the presence of exogenous S1P. Our study also demonstrated that S1P exerted its protective effect through the S1P1 receptor. Taken together, these results show that Sphk2/S1P has an important role to play in the survival of the dopaminergic neurons, in the pathogenesis of PD.

Molecular Mechanism Underlying PD Pathogenesis-A Possible Role for Sphingosine Kinases and Sphingosine-1-Phosphate

Parkinson’s disease is a neurodegenerative disorder that results in the degeneration of dopaminergic neurons in the substantia nigra (SNc) of the midbrain. Understanding the molecular mechanisms underlying the cause of Parkinson’s disease (PD) has attracted the attention of many researchers in the last few decades. In spite of the recent technical advances in the field of neuroscience, the complete pathophysiology of PD is not fully understood. Dysregulated sphingolipid metabolism has been shown to underlie the pathophysiology of many neurodegenerative disorders. However, the role of these sphingolipids in the pathophysiology of PD is still not known. This paper focuses on the, metabolic pathways that are involved in PD pathogenesis with emphasis on sphingolipids and the possible role played by them in the pathogenesis of PD.

Dysregulated Sphingolipid Metabolism in Endometriosis

In endometriosis, the establishment and subsistence of ectopic lesions outside the endometrium suggest an altered cellular state for pathological hyperplasia. Sphingolipids are bioactive compounds, and their biosynthesis and metabolism modulate a range of cellular processes including proliferation, migration and apoptosis. We demonstrate that aberrations in sphingolipid metabolism occur in women with endometriosis. Targeted mass spectrometry on >120 sphingolipids were measured in the sera (n = 62), peritoneal fluid (n = 63), and endometrial tissue (n = 14) of women with and without endometriosis. Quantitative RT-PCR and immunohistochemistry were performed on endometrial tissues determine the expression levels of sphingolipid enzymes. Sphingolipidomics identified the in vivo accumulation of numerous sphingolipids, including the functionally antagonistic glucosylceramides and ceramides in the serum and PF of women with endometriosis. We found upregulation of specific sphingolipid enzymes, namely sphingomyelin synthase 1 (SMS1), sphingomyelinase 3 (SMPD3), and glucosylceramide synthase (GCS) in the endometrium of endometriotic women with corresponding increased GlcCer, decreased sphingomyelin levels, and decreased apoptosis in the endometrium. Our sphingolipidomics approach provided evidence of altered sphingolipid metabolism flux in serum, peritoneal fluid, and endometrial tissue in women with endometriosis. The results provide new information on how sphingolipids and eutopic endometrium may contribute to the pathophysiology of endometriosis. The results also have implications for the use of sphingolipids as potential biomarkers.

Ceramide Transfer Protein Deficiency Compromises Organelle Function and Leads to Senescence in Primary Cells

Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum (ER) to the Golgi complex. Its deficiency in mouse leads to embryonic death at E11.5. CERT deficient embryos die from cardiac failure due to defective organogenesis, but not due to ceramide induced apoptotic or necrotic cell death. In the current study we examined the effect of CERT deficiency in a primary cell line, namely, mouse embryonic fibroblasts (MEFs). We show that in MEFs, unlike in mutant embryos, lack of CERT does not lead to increased ceramide but causes an accumulation of hexosylceramides. Nevertheless, the defects due to defective sphingolipid metabolism that ensue, when ceramide fails to be trafficked from ER to the Golgi complex, compromise the viability of the cell. Therefore, MEFs display an incipient ER stress. While we observe that ceramide trafficking from ER to the Golgi complex is compromised, the forward transport of VSVG-GFP protein is unhindered from ER to Golgi complex to the plasma membrane. However, retrograde trafficking of the plasma membrane-associated cholera toxin B to the Golgi complex is reduced. The dysregulated sphingolipid metabolism also leads to increased mitochondrial hexosylceramide. The mitochondrial functions are also compromised in mutant MEFs since they have reduced ATP levels, have increased reactive oxygen species, and show increased glutathione reductase activity. Live-cell imaging shows that the mutant mitochondria exhibit reduced fission and fusion events. The mitochondrial dysfunction leads to an increased mitophagy in the CERT mutant MEFs. The compromised organelle function compromise cell viability and results in premature senescence of these MEFs.

Targeting glucosylceramide synthase synergizes with C6-ceramide nanoliposomes to induce apoptosis in natural killer cell leukemia

Natural killer (NK) cell leukemia is characterized by clonal expansion of CD3 − NK cells and comprises both chronic and aggressive forms. Currently no effective treatment exists, thus providing a need for identification of novel therapeutics. Lipidomic studies revealed a dysregulated sphingolipid metabolism as evidenced by decreased levels of overall ceramide species and increased levels of cerebrosides in leukemic NK cells, concomitant with increased glucosylceramide synthase (GCS) expression. GCS, a key enzyme of this pathway, neutralizes pro-apoptotic ceramide by transfer of a uridine diphosphate (UDP)-glucose. Thus, we treated both rat and human leukemic NK cells in combination with: (1) exogenous C6-ceramide nanoliposomes in order to target mitochondria and increase physiological pro-apoptotic levels of long chain ceramide, and (2) 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of GCS. Co-administration of C6-ceramide nanoliposomes and PPMP elicited an increase in endogenous long-chain ceramide species, which led to cellular apoptosis in a synergistic manner via the mitochondrial intrinsic cell death pathway in leukemic NK cells.

Orm family proteins mediate sphingolipid homeostasis.

Despite the essential roles of sphingolipids both as structural components of membranes and critical signaling molecules, we have a limited understanding of how cells sense and regulate their levels. Our recent studies reveal the function in sphingolipid metabolism of the ORM genes (known as ORMDL genes in humans)-a conserved gene family that includes ORMDL3, which has recently been identified as a potential risk factor for childhood asthma. Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism. More recently, we have identified the regulatory kinase responsible for ORM phosphorylation in response to changes in lipid metabolism and have explored ORM regulation in mammalian systems. Our work identifies the Orm proteins as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma.

Orm family proteins mediate sphingolipid homeostasis.

Despite the essential roles of sphingolipids as both structural components of membranes and critical signalling molecules, we have a limited understanding of how cells sense and regulate their levels. Here we reveal the function in sphingolipid metabolism of the ORM/ORMDL genes, a conserved gene family that includes ORMDL3, which has recently been identified as a potential risk factor for childhood asthma. Starting from an unbiased functional genomic approach, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism. Our work identifies the Orm proteins as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma.

Neuronal Ceroid Lipofuscinosis: A Common Pathway?

The neuronal ceroid lipofuscinoses are pediatric neurodegenerative diseases with common clinical features. Of the nine clinical variants (CLN1-CLN9), six have been genetically identified. Most variants manifest cell death and dysregulated sphingolipid metabolism, suggesting the proteins defective in these disorders may interact along one pathway. NCL patient-derived cell lines exhibit cell growth and apoptotic defects that reverse following transfection with the wild-type gene. The membrane-bound proteins CLN3, CLN6, and CLN8 complement each other, as do CLN1 and CLN2 proteins, with respect to growth and apoptosis. The CLN2 protein also corrects growth and apoptosis in CLN3-, CLN6-, and CLN8-deficient cell lines. Neither CLN1-deficient nor CLN2-deficient growth defects are corrected by CLN3, CLN6, and CLN8 proteins. CLN2, CLN3, CLN6, and CLN8 proteins co-immunoprecipitate and co-localize to early and/or recycling endosomes and lipid rafts. Additionally, CLN2p and CLN1p co-immunoprecipitate. The work presented supports interactions between NCL proteins occurring at multiple points along one pathway.