Ceramide Degradation Research Articles

Alkaline ceramidase 1-mediated platelet ceramide catabolism mitigates vascular inflammation and abdominal aortic aneurysm formation.

Abdominal aortic aneurysm (AAA) is a highly lethal vascular disease. The role of platelets in AAA remains incompletely understood. Here we show that platelet ceramides, rather than other phospholipids, were elevated in an angiotensin II (AngII)-induced AAA murine model and in patients with AAA by using targeted lipidomic analysis. Among key ceramide metabolism enzymes, alkaline ceramidase 1 (Acer1) hydrolyzing ceramides were exclusively downregulated in AAA platelets. Platelet-specific Acer1 knockout mice were more susceptible to AAA upon AngII infusion without affecting hemostasis and thrombosis. Mechanistically, Acer1 deficiency in platelets facilitated platelet pro-inflammatory cytokine secretion as well as P-selectin-mediated circulating platelet-leukocyte aggregation and infiltration in aortic walls via the ceramide-p38 MAPK signaling axis. Of note, AngII repressed Acer1 expression in platelets by decreasing HuR-dependent mRNA stability. In conclusion, Acer1-mediated ceramide degradation in platelets exhibited anti-inflammatory effects and ameliorated AAA formation, potentially serving as a therapeutic target for AAA and other inflammatory vascular diseases.

1173-P: Maternally Supplemented PQQ Targets Ceramide Metabolism in Offspring to Blunt Developmental Programming of NAFLD

Maternal obesity is a key risk factor for pediatric nonalcoholic fatty liver disease (NAFLD). Using a mouse model of Western-style diet (WD)-induced maternal obesity, we studied effects of supplementing dams with pyrroloquinoline quinone (PQQ; a potent dietary antioxidant) during gestation and lactation on hepatic lipid metabolism and fibrosis in offspring at postnatal day (PND) 14 and at 16 wks after a 4 wk WD challenge. Maternal PQQ (mWDPQQ) decreased neonatal hepatic triglycerides (30%, P=0.0012) and altered bioactive sphingolipids in adult offspring compared with those exposed to maternal WD (mWD), with no change in weight gain. mWDPQQ increased very long chain ceramides (VLCer; 219%, P=0.0002), associated with protection from fibrosis, concomitant with decreased expression of genes promoting ceramide degradation (Asah2, P=0.0014 and Acer3, P=0.02). Expression of Scd1, a desaturase regulating triglyceride biosynthesis, was attenuated in mWDPQQ offspring at PND14 (P=0.0009) and at 16 wks (P=0.0012), while expression of Elovl3, an enzyme promoting elongation of saturated fatty acids and ceramide production, was increased (P=0.0026). Comparative pathway analysis of RNASeq data showed enrichment of pathways in fatty acid metabolism, hepatic fibrosis and, unexpectedly, signaling through the aryl hydrocarbon receptor (AHR; a ligand-activated transcription factor regulating immune function and hepatoxicity). Chromatin IP in mWDPQQ liver showed increased AHR binding to the Cyp1a1 promoter, its canonical target, as compared with mWD, suggesting AHR signaling may be activated by mWDPQQ. AHR binding to the Scd1 promoter, a known AHR target, was increased, as was binding to Asah2 and Acer3, putative new AHR targets regulating long-chain ceramide degradation. These findings suggest that maternal PQQ has both short and long-term effects on blunting steatosis/fibrosis and promoting VLCer increase through novel AHR targets, protecting offspring from NAFLD. Disclosure A.Mandala: None. A.M.Teague: None. R.Janssen: None. N.Patil: None. A.D.Joshi: None. J.E.Friedman: None. K.R.Jonscher: None. Funding National Institutes of Health (1R01DK121951)

Genetic ablation of Saposin-D in Krabbe disease eliminates psychosine accumulation but does not significantly improve demyelination.

Krabbe disease is an inherited demyelinating disease caused by a genetic deficiency of the lysosomal enzyme galactosylceramide (GalCer) β-galactosidase (GALC). The Twitcher (Twi) mouse is a naturally occurring, genetically and enzymatically authentic mouse model that mimics infantile-onset Krabbe disease. The major substrate for GALC is the myelin lipid GalCer. However, the pathogenesis of Krabbe disease has long been explained by the accumulation of psychosine, a lyso-derivative of GalCer. Two metabolic pathways have been proposed for the accumulation of psychosine: a synthetic pathway in which galactose is transferred to sphingosine and a degradation pathway in which GalCer is deacylated by acid ceramidase (ACDase). Saposin-D (Sap-D) is essential for the degradation of ceramide by ACDase in lysosome. In this study, we generated Twi mice with a Sap-D deficiency (Twi/Sap-D KO), which are genetically deficient in both GALC and Sap-D and found that very little psychosine accumulated in the CNS or PNS of the mouse. As expected, demyelination with the infiltration of multinucleated macrophages (globoid cells) characteristic of Krabbe disease was milder in Twi/Sap-D KO mice than in Twi mice both in the CNS and PNS during the early disease stage. However, at the later disease stage, qualitatively and quantitatively comparable demyelination occurred in Twi/Sap-D KO mice, particularly in the PNS, and the lifespans of Twi/Sap-D KO mice were even shorter than that of Twi mice. Bone marrow-derived macrophages from both Twi and Twi/Sap-D KO mice produced significant amounts of TNF-α upon exposure to GalCer and were transformed into globoid cells. These results indicate that psychosine in Krabbe disease is mainly produced via the deacylation of GalCer by ACDase. The demyelination observed in Twi/Sap-D KO mice may be mediated by a psychosine-independent, Sap-D-dependent mechanism. GalCer-induced activation of Sap-D-deficient macrophages/microglia may play an important role in the neuroinflammation and demyelination in Twi/Sap-D KO mice.

Skin inflammation and impaired adipogenesis in a mouse model of acid ceramidase deficiency.

Acid ceramidase catalyzes the degradation of ceramide into sphingosine and a free fatty acid. Acid ceramidase deficiency results in lipid accumulation in many tissues and leads to the development of Farber disease (FD). Typical manifestations of classical FD include formation of subcutaneous nodules and joint contractures as well as the development of a hoarse voice. Healthy skin depends on a unique lipid profile to form a barrier that confers protection from pathogens, prevents excessive water loss, and mediates cell-cell communication. Ceramides comprise ~50% of total epidermis lipids and regulate cutaneous homeostasis and inflammation. Abnormal skin development including visual skin lesions has been reported in FD patients, but a detailed study of FD skin has not been performed. We conducted a pathophysiological study of the skin in our mouse model of FD. We observed altered lipid composition in FD skin dominated by accumulation of all studied ceramide species and buildup of abnormal storage structures affecting mainly the dermis. A deficiency of acid ceramidase activity also led to the activation of inflammatory IL-6/JAK/signal transducer and activator of transcription 3 and noncanonical NF-κB signaling pathways. Last, we report reduced proliferation of FD mouse fibroblasts and adipose-derived stem/stromal cells (ASC) along with impaired differentiation of ASCs into mature adipocytes.

The acid ceramidase/ceramide axis controls parasitemia in Plasmodium yoelii-infected mice by regulating erythropoiesis.

Acid ceramidase (Ac) is part of the sphingolipid metabolism and responsible for the degradation of ceramide. As bioactive molecule, ceramide is involved in the regulation of many cellular processes. However, the impact of cell-intrinsic Ac activity and ceramide on the course of Plasmodium infection remains elusive. Here, we use Ac-deficient mice with ubiquitously increased ceramide levels to elucidate the role of endogenous Ac activity in a murine malaria model. Interestingly, ablation of Ac leads to alleviated parasitemia associated with decreased T cell responses in the early phase of Plasmodium yoelii infection. Mechanistically, we identified dysregulated erythropoiesis with reduced numbers of reticulocytes, the preferred host cells of P. yoelii, in Ac-deficient mice. Furthermore, we demonstrate that administration of the Ac inhibitor carmofur to wildtype mice has similar effects on P. yoelii infection and erythropoiesis. Notably, therapeutic carmofur treatment after manifestation of P. yoelii infection is efficient in reducing parasitemia. Hence, our results provide evidence for the involvement of Ac and ceramide in controlling P. yoelii infection by regulating red blood cell development.

Ceramide contributes to pathogenesis and may be targeted for therapy in VCP inclusion body myopathy.

Knock-in homozygote VCPR155H/R155H mutant mice are a lethal model of valosin-containing protein (VCP)-associated inclusion body myopathy associated with Paget disease of bone, frontotemporal dementia and amyotrophic lateral sclerosis. Ceramide (d18:1/16:0) levels are elevated in skeletal muscle of the mutant mice, compared to wild-type controls. Moreover, exposure to a lipid-enriched diet reverses lethality, improves myopathy and normalizes ceramide levels in these mutant mice, suggesting that dysfunctions in lipid-derived signaling are critical to disease pathogenesis. Here, we investigated the potential role of ceramide in VCP disease using pharmacological agents that manipulate the ceramide levels in myoblast cultures from VCP mutant mice and VCP patients. Myoblasts from wild-type, VCPR155H/+ and VCPR155H/R155H mice, as well as patient-induced pluripotent stem cells (iPSCs), were treated with an inhibitor of ceramide degradation to increase ceramide via acid ceramidase (ARN082) for proof of principle. Three chemically distinct inhibitors of ceramide biosynthesis via serine palmitoyl-CoA transferase (L-cycloserine, myriocin or ARN14494) were used as a therapeutic strategy to reduce ceramide in myoblasts. Acid ceramidase inhibitor, ARN082, elevated cellular ceramide levels and concomitantly enhanced pathology. Conversely, inhibitors of ceramide biosynthesis L-cycloserine, myriocin and ARN14494 reduced ceramide production. The results point to ceramide-mediated signaling as a key contributor to pathogenesis in VCP disease and suggest that manipulating this pathway by blocking ceramide biosynthesis might exert beneficial effects in patients with this condition. The ceramide pathway appears to be critical in VCP pathogenesis, and small-molecule inhibitors of ceramide biosynthesis might provide therapeutic benefits in VCP and related neurodegenerative diseases.

Neutral ceramidase is a marker for cognitive performance in rats and monkeys

BackgroundCeramides are lipid molecules determining cell integrity and intercellular signaling, and thus, involved in the pathogenesis of several psychiatric and neurodegenerative disorders. However, little is known about the role of particular enzymes of the ceramide metabolism in the mechanisms of normal behavioral plasticity. Here, we studied the contribution of neutral ceramidase (NC), one of the main enzymes mediating ceramide degradation, in the mechanisms of learning and memory in rats and non-human primates.MethodsNaïve Wistar rats and black tufted-ear marmosets (Callithrix penicillata) were tested in several tests for short- and long-term memory and then divided into groups with various memory performance. The activities of NC and acid ceramidase (AC) were measured in these animals. Additionally, anxiety and depression-like behavior and brain levels of monoamines were assessed in the rats.ResultsWe observed a predictive role of NC activity in the blood serum for superior performance of long-term object memory tasks in both species. A brain area analysis suggested that high NC activity in the ventral mesencephalon (VM) predicts better short-term memory performance in rats. High NC activity in the VM was also associated with worse long-term object memory, which might be mediated by an enhanced depression-like state and a monoaminergic imbalance.ConclusionsAltogether, these data suggest a role for NC in short- and long-term memory of various mammalian species. Serum activity of NC may possess a predictive role in the assessing the performance of certain types of memory.

CrossTalk proposal: Intramuscular lipid accumulation causes insulin resistance.

CrossTalk proposal: Intramuscular lipid accumulation causes insulin resistance.

Role of Ceramidases in Sphingolipid Metabolism and Human Diseases.

Human pathologies such as Alzheimer’s disease, type 2 diabetes-induced insulin resistance, cancer, and cardiovascular diseases have altered lipid homeostasis. Among these imbalanced lipids, the bioactive sphingolipids ceramide and sphingosine-1 phosphate (S1P) are pivotal in the pathophysiology of these diseases. Several enzymes within the sphingolipid pathway contribute to the homeostasis of ceramide and S1P. Ceramidase is key in the degradation of ceramide into sphingosine and free fatty acids. In humans, five different ceramidases are known—acid ceramidase, neutral ceramidase, and alkaline ceramidase 1, 2, and 3—which are encoded by five different genes (ASAH1, ASAH2, ACER1, ACER2, and ACER3, respectively). Notably, the neutral ceramidase N-acylsphingosine amidohydrolase 2 (ASAH2) shows considerable differences between humans and animals in terms of tissue expression levels. Besides, the subcellular localization of ASAH2 remains controversial. In this review, we sum up the results obtained for identifying gene divergence, structure, subcellular localization, and manipulating factors and address the role of ASAH2 along with other ceramidases in human diseases.

Ca2+ Signaling and Barrier Function of Lung Microvascular Endothelial Cells are Modulated by Mesenchymal Stromal Cell Microparticles

IntroductionAcute respiratory distress syndrome (ARDS) is a fatal condition characterized by hyperinflammation and pulmonary microvascular leak. With no pharmacological cure, treatment remains only supportive. Mesenchymal stromal cells (MSCs) have recently generated excitement as a potential therapy, presumably functioning through paracrine mechanisms which may rely on the release of extracellular vesicles like microparticles (MPs). However, the mechanism by which MPs modulate the pathology of ARDS is poorly understood yet may pave the way to develop cell‐free cell therapeutics.ObjectiveTo identify the mechanism(s) by which MSC derived MPs enhance pulmonary microvascular barrier function.MethodsMPs were purified from conditioned medium produced by MSCs either stimulated with Ca2+ ionophore or unstimulated. Monolayers of primary human pulmonary microvascular endothelial cells (HPMECs) were injured with thrombin to induce barrier disruption, measured by transendothelial electrical resistance (TEER), mimicking lung microvascular leak as observed in ARDS. MPs were administered to treat thrombin‐injured HPMECs and changes in intracellular Ca2+ concentration ([Ca2+]i) were determined by ratiometric imaging of Fura‐2.ResultsThrombin administered to HPMECs caused an initial, brief spike in [Ca2+]i followed by a second phase characterized as a prolonged, gradual increase in [Ca2+]i. Following the thrombin‐induced [Ca2+]i spike, treatment with MPs from unstimulated MSCs eliminated the second phase and caused a sustained, decrease in [Ca2+]i below baseline. In parallel, MPs from unstimulated MSCs enhanced TEER recovery and VE‐cadherin integrity in intercellular junctions of HPMECs following thrombin‐induced permeability. Conversely, treatment with MPs from Ca2+ ionophore stimulated MSCs amplified the second phase of the [Ca2+]i response to thrombin and prevented recovery of HPMEC barrier function. HPLC‐MS revealed MPs from unstimulated MSCs had lower ceramide and higher sphingosine‐1‐phosphate (S1P) content than MPs from Ca2+ ionophore stimulated MSCs. S1P degradation by S1P lyase or blockade of the S1P receptor 1 attenuated the barrier‐protective effect of MPs from unstimulated MSCs, while ceramide degradation by neutral ceramidase improved barrier recovery following treatment with MPs from Ca2+ ionophore stimulated MSCs.ConclusionMPs from unstimulated MSCs therapeutically enhance lung capillary barrier function, presumably by attenuating endothelial [Ca2+]i responses by resetting the ceramide/S1P rheostat. These findings provide a mechanistic basis for development and optimization/enrichment of MSC derived MPs as a promising cell‐free cell therapy for ARDS.Support or Funding InformationThis work was supported by the Ontario Research Fund and Canadian Institutes of Health Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel glucosylceramide synthase inhibitor based prodrug copolymer micelles for delivery of anticancer agents

In order to improve the efficacy of chemotherapy for cancers, we have developed a novel prodrug micellar formulation to co-deliver ceramide-generating anticancer agents and ceramide degradation inhibitor (PPMP). The prodrug nanocarrier is based on a well-defined POEG-b-PPPMP diblock copolymer. The hydrophilic block of POEG-b-PPPMP is POEG, and the hydrophobic block is composed of a number of PPMP units, which could work synergistically with loaded anticancer drugs. POEG-b-PPPMP was readily synthesized via a one-step reversible addition-fragment transfer (RAFT) polymerization from a PPMP monomer. The newly synthesized polymers were self-assembled into micelles and served as a carrier for several hydrophobic anticancer drugs including DOX, PTX and C6-ceramide. POEG-b-PPPMP prodrug polymer exhibited intrinsic antitumor activity in vitro and in vivo. In addition, POEG-b-PPPMP prodrug polymer was comparable to free PPMP in selectively enhancing the production of pro-apoptotic ceramide species as well as down-regulating the mRNA expression of GCS. DOX-loaded POEG-b-PPPMP micelles exhibited an excellent stability of 42 days at 4 °C. Moreover, DOX loaded in POEG-b-PPPMP micelles showed higher levels of cytotoxicity than DOX loaded in a pharmacologically inert polymer (POEG-b-POM) and Doxil formulation in several tumor cell lines. Consistently, in a 4T1.2 murine breast cancer model, the tumor inhibition followed the order of DOX/POEG-b-PPPMP > DOX/POEG-b-POM ≥ Doxil > POEG-b-PPPMP > POEG-b-POM. Our data suggest that POEG-b-PPPMP micelles are a promising dual-functional carrier that warrants more studies in the future.

Mesenchymal Stromal Cell Microparticles Enhance Lung Endothelial Barrier Through CD44 and the S1P/ceramide Rheostat

IntroductionAcute respiratory distress syndrome (ARDS) is a fatal condition characterized by hyperinflammation and pulmonary microvascular leak. Treatment is only supportive with no pharmacological cure. Recently, mesenchymal stromal/stem cells (MSCs) have emerged as potential therapy, but greater knowledge on their mechanism(s) of action could yield development of cell‐free therapeutics.ObjectiveTo determine if MSC‐produced microparticles (MPs) ameliorate endothelial barrier disruption and identify their biomolecular contents responsible for such therapeutic effect.MethodsTo re‐capitulate lung capillary leak in ARDS, primary human pulmonary microvascular endothelial cell (HPMEC) monolayers grown on Transwell inserts were injured with thrombin to induce barrier disruption as measured by transendothelial electrical resistance (TEER). MPs were purified out of conditioned medium produced by MSCs either stimulated with Ca2+ ionophore or unstimulated. MPs were administered to treat thrombin‐injured HPMEC monolayers; surface markers were compared by flow cytometry and lipid content analyzed by HPLC‐MS.ResultsMPs from unstimulated MSCs enhanced HPMEC barrier function and TEER recovery after thrombin injury, while MPs from stimulated MSCs were injurious. Stimulated MSCs produced MPs with significantly less surface CD44. Antibody blockade of CD44 on unstimulated MPs eliminated their therapeutic effect on HPMEC monolayers. Moreover, stimulated MPs had significantly more total ceramides and less sphingosine‐1‐phosphate (S1P) than unstimulated MPs. Ceramide degradation by neutral ceramidase blunted the injurious effect of stimulated MPs. The therapeutic function of unstimulated MPs was eliminated with ceramide over‐production by acid sphingomyelinase or S1P degradation by S1P lyase.ConclusionThese results show MSC‐produced MPs were therapeutic after lung capillary endothelial barrier disruption. Beneficial effects were associated with surface CD44 and greater ratio of S1P:ceramide content. This knowledge provides a mechanistic basis for development of MSC‐based cell‐free therapeutics.Support or Funding InformationThis work was supported by Ontario Research Fund and Canadian Institutes of Health Research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protective Effects of Gomisin N against Hepatic Cannabinoid Type 1 Receptor-Induced Insulin Resistance and Gluconeogenesis.

Activation of the hepatic cannabinoid type 1 receptor (CB1R) induces insulin resistance and gluconeogenesis via endoplasmic reticulum (ER) stress, thereby contributing to hyperglycemia. Gomisin N (GN) is a phytochemical derived from Schisandra chinensis. In the current study, we investigated the inhibitory effects of GN on hepatic CB1R-mediated insulin resistance and gluconeogenesis in 2-arachidonoylglycerol (AG; an agonist of CB1R)-treated HepG2 cells and in high-fat diet (HFD)-induced obese mice. Treatment with 2-AG induced the expression of ER stress markers, serine/threonine phosphatase PHLPP1, Lipin1, and ceramide synthesis genes, but reduced the expression of ceramide degradation genes in HepG2 cells. However, GN reversed 2-AG-mediated effects and improved the 2-AG-mediated impairment of insulin signaling. Furthermore, GN inhibited 2-AG-induced intracellular triglyceride accumulation and glucose production in HepG2 cells by downregulation of lipogenesis and gluconeogenesis genes, respectively. In vivo, GN administration to HFD obese mice reduced the HFD-induced increase in fasting blood glucose and insulin levels, which was accompanied with downregulation of HFD-induced expression of CB1R, ER stress markers, ceramide synthesis gene, and gluconeogenesis genes in the livers of HFD obese mice. These findings demonstrate that GN protects against hepatic CB1-mediated impairment of insulin signaling and gluconeogenesis, thereby contributing to the amelioration of hyperglycemia.

Neutral ceramidase activity inhibition is involved in palmitate-induced apoptosis in INS-1 cells.

Neutral ceramidase (NCDase) is a class of ceramidases, a key enzyme in ceramide degradation. Recently, it was observed that NCDase activity was suppressed by saturated fatty acids to increase ceramide content in rat muscle. However, little is known about its changes in activity and roles in palmitate (Palm)-induced lipotoxicity in pancreatic β cells. Here, we demonstrated that Palm treatment significantly down-regulated NCDase activity, mRNA and protein levels in rat INS-1 cells. In addition, Palm caused a significant accumulation of ceramide, while SPH level remained unchanged, suggesting that inhibition of NCDase activity led to no change of SPH level after treatment with Palm for 24 h. Furthermore, NCDase overexpression significantly reduced Palm-induced apoptosis in INS-1 cells. Conversely, NCDase siRNA knockdown markedly exacerbated Palm-induced apoptosis. In conclusion, Palm treatment suppressed the activity of NCDase and down-regulated its mRNA and protein expression. Furthermore, NCDase inhibition was involved in Palm-induced apoptosis by blocking ceramide degradation in INS-1 cells.

Spinal muscular atrophy associated with progressive myoclonus epilepsy

A rare syndrome characterized by lower motor neuron disease associated with progressive myoclonic epilepsy, referred to as "spinal muscular atrophy associated with progressive myoclonic epilepsy" (SMA-PME), has been described in childhood and is inherited as an autosomal recessive trait. SMA-PME is caused by mutation in the ASAH1 gene encoding acid ceramidase. Ceramide and the metabolites participate in various cellular events as lipid mediators. The catabolism of ceramide in mammals occurs in lysosomes through the activity of ceramidase. Three different ceramidases (acid, neutral and alkaline) have been identified and appear to play distinct roles in sphingolipid metabolism. The enzymatic activity of acid ceramidase is deficient in two rare inherited disorders; Farber disease and SMA-PME. Farber disease is a very rare and severe autosomal recessive condition with a distinct clinical phenotype. The marked difference in disease manifestations may explain why Farber and SMA-PME diseases were not previously suspected to be allelic conditions. The precise molecular mechanism underlying the phenotypic differences remains to be clarified. Recently, a condition with mutation in CERS1, the gene encoding ceramide synthase 1, has been identified as a novel form of PME. This finding underlies the essential role of enzymes regulating either the synthesis (CERS1) or degradation (ASAH1) of ceramide, and the link between defects in ceramide metabolism and PME.

Abstract 2895: Radiation-induced alternative splicing as detected in total and polysome-bound mRNA in glioblastoma stem-like cells

Abstract The vast majority of human genes undergo alternative splicing, which is dependent on tissue type, developmental stage, and environmental context. Given that alternative splicing regulates gene expression and is a major source of diversity within the proteome, we used RNAseq to determine the effects of ionizing radiation on the generation of alternative transcripts in the human glioblastoma stem-like cell line NSC11. For these studies, total cellular mRNA, reflecting the transcriptome, and polysome-bound mRNA, reflecting the translatome, were collected from control and irradiated (2Gy, 1h) NSC11 cells and subjected to RNAseq to identify all transcripts; SpliceSeq was then used to visualize and quantitate splice variations induced by radiation. We found that after irradiation 122 genes were differentially spliced in the transcriptome, which indicates that radiation induces alternative splicing, while 357 were differentially spliced in the translatome suggesting that radiation also increases the translation of specific alternative transcripts. However, 33 genes were in common between the transcriptome and translatome suggesting that radiation increases the level and translation of these alternative transcripts. IPA analyses of the radiation-induced alternative transcripts in the translatome identified DNA Double-Strand Break Repair by Homologous Recombination, Ceramide Degradation, and Sphingosine and Sphingosine-1-phospahate Metabolism as the top 3 canonical pathways enriched in this dataset. The top 3 molecular and cellular functions in this analysis were Cellular Assembly and Organization, DNA Replication, Recombination, and Repair, and Cell Cycle. The same analysis of alternative transcripts in the transcriptome did not show an enhancement of DNA damage and repair networks after radiation, suggesting that it is the polysome loading of these transcripts that is changed after irradiation. Further analyses of alternative transcripts in the translatome using EnrichR demonstrated that the top 3 pathways, analyzed using the Reactome 2015 curated pathway database, were Homologous Recombination Repair of Replication-Independent Double-Strand Breaks, Homologous Recombination Repair, and Double-Strand Break Repair. Finally, GeneCodis was used to analyze the biological processes enhanced in the radiation-induced translatome. The top 3 biological processes were Regulation of Transcription, DNA-Dependent, Negative Regulation of Transcription, DNA-Dependent, and Double-Strand Break Repair. These results indicate that radiation affects alternative splicing at both the level of mRNA abundance and translation and suggest that these processes may play a role in radiation-induced translational control of gene expression. Citation Format: Amy Wahba, Michael C. Ryan, Uma Shankavaram, Kevin Camphausen, Philip J. Tofilon. Radiation-induced alternative splicing as detected in total and polysome-bound mRNA in glioblastoma stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2895.

ASAH1 variant causing a mild SMA phenotype with no myoclonic epilepsy: a clinical, biochemical and molecular study.

ASAH1 gene encodes for acid ceramidase that is involved in the degradation of ceramide into sphingosine and free fatty acids within lysosomes. ASAH1 variants cause both the severe and early-onset Farber disease and rare cases of spinal muscular atrophy (SMA) with progressive myoclonic epilepsy (SMA-PME), phenotypically characterized by childhood onset of proximal muscle weakness and atrophy due to spinal motor neuron degeneration followed by occurrence of severe and intractable myoclonic seizures and death in the teenage years. We studied two subjects, a 30-year-old pregnant woman and her 17-year-old sister, affected with a very slowly progressive non-5q SMA since childhood. No history of seizures or myoclonus has been reported and EEG was unremarkable. The molecular study of ASAH1 gene showed the presence of the homozygote nucleotide variation c.124A>G (r.124a>g) that causes the amino acid substitution p.Thr42Ala. Biochemical evaluation of cultured fibroblasts showed both reduction in ceramidase activity and accumulation of ceramide compared with the normal control. This study describes for the first time the association between ASAH1 variants and an adult SMA phenotype with no myoclonic epilepsy nor death in early age, thus expanding the phenotypic spectrum of ASAH1-related SMA. ASAH1 molecular analysis should be considered in the diagnostic testing of non-5q adult SMA patients.

Atom based 3D-QSAR studies on 2,4-dioxopyrimidine-1-carboxamide analogs: Validation of experimental inhibitory potencies towards acid ceramidase

Ceramide (Cer), the central lipid molecule in sphingolipid biosynthesis and degradation, which plays a key role in sphingolipid signaling, induces cell differentiation and apoptosis. Cellular degradation of ceramide to sphingosine is catalyzed by a family of ceramidases (CDases). Pharmacological inhibition of ceramidases and more particularly, acid ceramidase (aCDase) is suggestive of a chemotherapeutic approach as it increases the cellular concentration of ceramide inducing apoptosis. In the present report, we have utilized atom-based 3D-QSAR method to analyze the structural aspects on a series of 2,4-dioxopyrimidine-1-carboxamide (carmofur) derivatives as potent inhibitors of aCDase. In this approach the experimental dataset was divided into training (83%) and test (17%) sets and the best model was chosen based on randomized trial distributions consisting of five compounds in a test set with a wide range of activity profile and superior values of statistical parameters such as Q2 and R2 values. The reported experimental results by Piomelli and co-workers on the inhibition of aCDase by the carmofur derivatives were correlated using robust 3D-QSAR as well as docking methods. With careful structure–activity correlation studies the carmofur analogs were classified into four sub-categories (Set 1–4) to understand the effect of each structural features separately. This approach led us to short-list most active carmofur derivatives such as compounds 26, 30 and 32 with the incorporation of more than one structural features in a single molecule. However, the inhibition potency might further be enhanced by designing compound 33 upon the incorporation of all features in a single compound. Compound 33 that was missing in the experimental study by Piomelli and co-workers (J. Med. Chem. 2013, 56, 3518), could be identified using 3D-QSAR studies. Moreover, the importance of structural features in lead inhibitors such as 26, 30 and 32 along with 33 was further justified by their efficient molecular interactions at the active site of homology modeled protein human N-acyl ethanolamine hydrolyzing acid amidase (hNAAA) as evidenced by molecular docking study. Furthermore, efficient molecular interaction of some representative inhibitors with hNAAA led to the understanding that hNAAA could be a possible alternative of aCDase for developing potent inhibitors.

Inhibitors of Ceramidases

The topic of ceramidases has experienced an enormous boost during the last few years. Ceramidases catalyze the degradation of ceramide to sphingosine and fatty acids. Ceramide is not only the central hub of sphingolipid biosynthesis and degradation, it is also a key molecule in sphingolipid signaling, promoting differentiation or apoptosis. Acid ceramidase inhibition sensitizes certain types of cancer to chemo- and radio-therapy and this is suggestive of a role of acid ceramidase inhibitors as chemo-sensitizers which can act synergistically with chemo-therapeutic drugs. In this review, we summarize the development of ceramide analogues as first-generation ceramidase inhibitors together with data on their activity in cells and disease models. Furthermore, we describe the recent developments that have led to highly potent second-generation ceramidase inhibitors that act at nanomolar concentrations. In the third part, various assays of ceramidases are described and their relevance for accurately measuring ceramidase activities and for the development of novel inhibitors is highlighted. Besides potential clinical implications, the recent improvements in ceramidase inhibition and assaying may help to better understand the mechanisms of ceramide biology.

Targeted Induction of Ceramide Degradation Leads to Improved Systemic Metabolism and Reduced Hepatic Steatosis

Sphingolipids have garnered attention for their role in insulin resistance and lipotoxic cell death. We have developed transgenic mice inducibly expressing acid ceramidase that display a reduction in ceramides in adult mouse tissues. Hepatic overexpression of acid ceramidase prevents hepatic steatosis and prompts improvements in insulin action in liver and adipose tissue upon exposure to high-fat diet. Conversely, overexpression of acid ceramidase within adipose tissue also prevents hepatic steatosis and systemic insulin resistance. Induction of ceramidase activity in either tissue promotes a lowering of hepatic ceramides and reduced activation of the ceramide-activated protein kinase C isoform PKCζ, though the induction of ceramidase activity in the adipocyte prompts more rapid resolution of hepatic steatosis than overexpression of the enzyme directly in the liver. Collectively, our observations suggest the existence of a rapidly acting "cross-talk" between liver and adipose tissue sphingolipids, critically regulating glucose metabolism and hepatic lipid uptake.