S-Adenosyl-l-methionine restores brain mitochondrial membrane fluidity and GSH content improving Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by impaired motor coordination due to neurological defects and cerebellar dysfunction caused by the accumulation of cholesterol in endolysosomes. Besides the increase in lysosomal cholesterol, mitochondria are also enriched in cholesterol, which leads to decreased membrane fluidity, impaired mitochondrial function and loss of GSH, and has been shown to contribute to the progression of NPC disease. S-Adenosyl-l-methionine (SAM) regulates membrane physical properties through the generation of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) methylation and functions as a GSH precursor by providing cysteine in the transsulfuration pathway. However, the role of SAM in NPC disease has not been investigated. Here we report that Npc1-/- mice exhibit decreased brain SAM levels but unchanged S-adenosyl-l-homocysteine content and lower expression of Mat2a. Brain mitochondria from Npc1-/- mice display decreased mitochondrial GSH levels and liquid chromatography-high resolution mass spectrometry analysis reveal a lower PC/PE ratio in mitochondria, contributing to increased mitochondrial membrane order. In vivo treatment of Npc1-/- mice with SAM restores SAM levels in mitochondria, resulting in increased PC/PE ratio, mitochondrial membrane fluidity and subsequent replenishment of mitochondrial GSH levels. In vivo SAM treatment improves the decline of locomotor activity, increases Purkinje cell survival in the cerebellum and extends the average and maximal life spam of Npc1-/- mice. These findings identify SAM as a potential therapeutic approach for the treatment of NPC disease.

Simultaneous Inhibition of Ceramide Hydrolysis and Glycosylation Synergizes to Corrupt Mitochondrial Respiration and Signal Caspase Driven Cell Death in Drug-Resistant Acute Myeloid Leukemia.

Acute myelogenous leukemia (AML), the most prevalent acute and aggressive leukemia diagnosed in adults, often recurs as a difficult-to-treat, chemotherapy-resistant disease. Because chemotherapy resistance is a major obstacle to successful treatment, novel therapeutic intervention is needed. Upregulated ceramide clearance via accelerated hydrolysis and glycosylation has been shown to be an element in chemotherapy-resistant AML, a problem considering the crucial role ceramide plays in eliciting apoptosis. Herein we employed agents that block ceramide clearance to determine if such a "reset" would be of therapeutic benefit. SACLAC was utilized to limit ceramide hydrolysis, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) was used to block the glycosylation route. The SACLAC D-threo-PDMP inhibitor combination was synergistically cytotoxic in drug-resistant, P-glycoprotein-expressing (P-gp) AML but not in wt, P-gp-poor cells. Interestingly, P-gp antagonists that can limit ceramide glycosylation via depression of glucosylceramide transit also synergized with SACLAC, suggesting a paradoxical role for P-gp in the implementation of cell death. Mechanistically, cell death was accompanied by a complete drop in ceramide glycosylation, concomitant, striking increases in all molecular species of ceramide, diminished sphingosine 1-phosphate levels, resounding declines in mitochondrial respiratory kinetics, altered Akt, pGSK-3ß, and Mcl-1 expression, and caspase activation. Although ceramide was generated in wt cells upon inhibitor exposure, mitochondrial respiration was not corrupted, suggestive of mitochondrial vulnerability in the drug-resistant phenotype, a potential therapeutic avenue. The inhibitor regimen showed efficacy in an in vivo model and in primary AML cells from patients. These results support the implementation of SL enzyme targeting to limit ceramide clearance as a therapeutic strategy in chemotherapy-resistant AML, inclusive of a novel indication for the use of P-gp antagonists.

High-throughput discovery of novel small-molecule inhibitors of acid Ceramidase.

Ceramide has a key role in the regulation of cellular senescence and apoptosis. As Ceramide levels are lowered by the action of acid ceramidase (AC), abnormally expressed in various cancers, the identification of AC inhibitors has attracted increasing interest. However, this finding has been mainly hampered by the lack of formats suitable for the screening of large libraries. We have overcome this drawback by adapting a fluorogenic assay to a 384-well plate format. The performance of this optimised platform has been proven by the screening a library of 4100 compounds. Our results show that the miniaturised platform is well suited for screening purposes and it led to the identification of several hits, that belong to different chemical classes and display potency ranges of 2-25 µM. The inhibitors also show selectivity over neutral ceramidase and retain activity in cells and can therefore serve as a basis for further chemical optimisation.

Methuosis Contributes to Jaspine-B-Induced Cell Death.

Methuosis is a type of programmed cell death in which the cytoplasm is occupied by fluid-filled vacuoles that originate from macropinosomes (cytoplasmic vacuolation). A few molecules have been reported to behave as methuosis inducers in cancer cell lines. Jaspine B (JB) is a natural anhydrous sphingolipid (SL) derivative reported to induce cytoplasmic vacuolation and cytotoxicity in several cancer cell lines. Here, we have investigated the mechanism and signalling pathways involved in the cytotoxicity induced by the natural sphingolipid Jaspine B (JB) in lung adenocarcinoma A549 cells, which harbor the G12S K-Ras mutant. The effect of JB on inducing cytoplasmic vacuolation and modifying cell viability was determined in A549 cells, as well as in mouse embryonic fibroblasts (MEF) lacking either the autophagy-related gene ATG5 or BAX/BAK genes. Apoptosis was analyzed by flow cytometry after annexin V/propidium iodide staining, in the presence and absence of z-VAD. Autophagy was monitored by LC3-II/GFP-LC3-II analysis, and autophagic flux experiments using protease inhibitors. Phase contrast, confocal, and transmission electron microscopy were used to monitor cytoplasmic vacuolation and the uptake of Lucifer yellow to assess macropinocyosis. We present evidence that cytoplasmic vacuolation and methuosis are involved in Jaspine B cytotoxicity over A549 cells and that activation of 5' AMP-activated protein kinase (AMPK) could be involved in Jaspine-B-induced vacuolation, independently of the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin complex 1 (PI3K/Akt/mTORC1) axis.

Discovery of deoxyceramide analogs as highly selective ACER3 inhibitors in live cells.

Acid (AC), neutral (NC) and alkaline ceramidase 3 (ACER3) are the most ubiquitous ceramidases and their therapeutic interest as targets in cancer diseases has been well sustained. This supports the importance of discovering potent and specific inhibitors for further use in combination therapies. Although several ceramidase inhibitors have been reported, most of them target AC and a few focus on NC. In contrast, well characterized ACER3 inhibitors are lacking. Here we report on the synthesis and screening of two series of 1-deoxy(dihydro)ceramide analogs on the three enzymes. Activity was determined using fluorogenic substrates in recombinant human NC (rhNC) and both lysates and intact cells enriched in each enzyme. None of the molecules elicited a remarkable AC inhibitory activity in either experimental setup, while using rhNC, several compounds of both series were active as non-competitive inhibitors with Ki values between 1 and 5 µM. However, a dramatic loss of potency occurred in NC-enriched cell lysates and no activity was elicited in intact cells. Interestingly, several compounds of Series 2 inhibited ACER3 dose-dependently in both cell lysates and intact cells with IC50's around 20 µM. In agreement with their activity in live cells, they provoked a significant increase in the amounts of ceramides. Overall, this study identifies highly selective ACER3 activity blockers in intact cells, opening the door to further medicinal chemistry efforts aimed at developing more potent and specific compounds.

The anti-cancer drug ABTL0812 induces ER stress-mediated cytotoxic autophagy by increasing dihydroceramide levels in cancer cells.

ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased DDIT3 and TRIB3 mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer.Abbreviations: 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ9-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response.

ß-Galactosylceramidase Promotes Melanoma Growth via Modulation of Ceramide Metabolism.

Disturbance of sphingolipid metabolism may represent a novel therapeutic target in metastatic melanoma, the most lethal form of skin cancer. ß-Galactosylceramidase (GALC) removes ß-galactose from galactosylceramide and other sphingolipids. In this study, we show that downregulation of galcb, a zebrafish ortholog of human GALC, affects melanoblast and melanocyte differentiation in zebrafish embryos, suggesting a possible role for GALC in melanoma. On this basis, the impact of GALC expression in murine B16-F10 and human A2058 melanoma cells was investigated following its silencing or upregulation. Galc knockdown hampered growth, motility, and invasive capacity of B16-F10 cells and their tumorigenic and metastatic activity when grafted in syngeneic mice or zebrafish embryos. Galc-silenced cells displayed altered sphingolipid metabolism and increased intracellular levels of ceramide, paralleled by a nonredundant upregulation of Smpd3, which encodes for the ceramide-generating enzyme neutral sphingomyelinase 2. Accordingly, GALC downregulation caused SMPD3 upregulation, increased ceramide levels, and inhibited the tumorigenic activity of human melanoma A2058 cells, whereas GALC upregulation exerted opposite effects. In concordance with information from melanoma database mining, RNAscope analysis demonstrated a progressive increase of GALC expression from common nevi to stage IV human melanoma samples that was paralleled by increases in microphthalmia transcription factor and tyrosinase immunoreactivity inversely related to SMPD3 and ceramide levels. Overall, these findings indicate that GALC may play an oncogenic role in melanoma by modulating the levels of intracellular ceramide, thus providing novel opportunities for melanoma therapy. SIGNIFICANCE: Data from zebrafish embryos, murine and human cell melanoma lines, and patient-derived tumor specimens indicate that ß-galactosylceramidase plays an oncogenic role in melanoma and may serve as a therapeutic target.

Ceramide Analogue SACLAC Modulates Sphingolipid Levels and MCL-1 Splicing to Induce Apoptosis in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a disease characterized by uncontrolled proliferation of immature myeloid cells in the blood and bone marrow. The 5-year survival rate is approximately 25%, and recent therapeutic developments have yielded little survival benefit. Therefore, there is an urgent need to identify novel therapeutic targets. We previously demonstrated that acid ceramidase (ASAH1, referred to as AC) is upregulated in AML and high AC activity correlates with poor patient survival. Here, we characterized a novel AC inhibitor, SACLAC, that significantly reduced the viability of AML cells with an EC50 of approximately 3 µmol/L across 30 human AML cell lines. Treatment of AML cell lines with SACLAC effectively blocked AC activity and induced a decrease in sphingosine 1-phosphate and a 2.5-fold increase in total ceramide levels. Mechanistically, we showed that SACLAC treatment led to reduced levels of splicing factor SF3B1 and alternative MCL-1 mRNA splicing in multiple human AML cell lines. This increased proapoptotic MCL-1S levels and contributed to SACLAC-induced apoptosis in AML cells. The apoptotic effects of SACLAC were attenuated by SF3B1 or MCL-1 overexpression and by selective knockdown of MCL-1S. Furthermore, AC knockdown and exogenous C16-ceramide supplementation induced similar changes in SF3B1 level and MCL-1S/L ratio. Finally, we demonstrated that SACLAC treatment leads to a 37% to 75% reduction in leukemic burden in two human AML xenograft mouse models. IMPLICATIONS: These data further emphasize AC as a therapeutic target in AML and define SACLAC as a potent inhibitor to be further optimized for future clinical development.

Chemotherapy selection pressure alters sphingolipid composition and mitochondrial bioenergetics in resistant HL-60 cells.

The combination of daunorubicin (dnr) and cytarabine (Ara-C) is a cornerstone of treatment for acute myelogenous leukemia (AML); resistance to these drugs is a major cause of treatment failure. Ceramide, a sphingolipid (SL), plays a critical role in cancer cell apoptosis in response to chemotherapy. Here, we investigated the effects of chemotherapy selection pressure with Ara-C and dnr on SL composition and enzyme activity in the AML cell line HL-60. Resistant cells, those selected for growth in Ara-C- and dnr-containing medium (HL-60/Ara-C and HL-60/dnr, respectively), demonstrated upregulated expression and activity of glucosylceramide synthase, acid ceramidase (AC), and sphingosine kinase 1 (SPHK1); were more resistant to ceramide than parental cells; and displayed sensitivity to inhibitors of SL metabolism. Lipidomic analysis revealed a general ceramide deficit and a profound upswing in levels of sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) in HL-60/dnr cells versus parental and HL-60/Ara-C cells. Both chemotherapy-selected cells also exhibited comprehensive upregulations in mitochondrial biogenesis consistent with heightened reliance on oxidative phosphorylation, a property that was partially reversed by exposure to AC and SPHK1 inhibitors and that supports a role for the phosphorylation system in resistance. In summary, dnr and Ara-C selection pressure induces acute reductions in ceramide levels and large increases in S1P and C1P, concomitant with cell resilience bolstered by enhanced mitochondrial remodeling. Thus, strategic control of ceramide metabolism and further research to define mitochondrial perturbations that accompany the drug-resistant phenotype offer new opportunities for developing therapies that regulate cancer growth.

Activity-Based Imaging of Acid Ceramidase in Living Cells.

Acid ceramidase (AC) hydrolyzes ceramides into sphingoid bases and fatty acids. The enzyme is overexpressed in several types of cancer and Alzheimer's disease, and its genetic defect causes different incurable disorders. The availability of a method for the specific visualization of catalytically active AC in intracellular compartments is crucial for diagnosis and follow-up of therapeutic strategies in diseases linked to altered AC activity. This work was undertaken to develop activity-based probes for the detection of AC. Several analogues of the AC inhibitor SABRAC were synthesized and found to act as very potent (two-digit nM range) irreversible AC inhibitors by reaction with the active site Cys143. Detection of active AC in cell-free systems was achieved either by using fluorescent SABRAC analogues or by click chemistry with an azide-substituted analogue. The compound affording the best features allowed the unprecedented labeling of active AC in living cells.

Ovarian Hormone-Dependent Effects of Dietary Lipids on APP/PS1 Mouse Brain.

The formation of senile plaques through amyloid-ß peptide (Aß) aggregation is a hallmark of Alzheimer's disease (AD). Irrespective of its actual role in the synaptic alterations and cognitive impairment associated with AD, different therapeutic approaches have been proposed to reduce plaque formation. In rodents, daily intake of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFAs) is required for neural development, and there is experimental and epidemiological evidence that their inclusion in the diet has positive effects on several neurodegenerative diseases. Similarly, estradiol appears to reduce senile plaque formation in primary mouse cell cultures, human cortical neurons and mouse AD models, and it prevents Aß toxicity in neural cell lines. We previously showed that differences in dietary n-6/n-3 LC-PUFAs ratios modify the lipid composition in the cerebral cortex of female mice and the levels of amyloid precursor protein (APP) in the brain. These effects depended in part on the presence of circulating estradiol. Here we explored whether this potentially synergistic action between diet and ovarian hormones may influence the progression of amyloidosis in an AD mouse model. Our results show that a diet with high n-3 LC-PUFA content, especially DHA (22:6n-3), reduces the hippocampal accumulation of Aß1 - 4 0, but not amyloid Aß1 - 42 in female APPswe/PS1 E9A mice, an effect that was counteracted by the loss of the ovaries and that depended on circulating estradiol. In addition, this interaction between dietary lipids and ovarian function also affects the composition of the brain lipidome as well as the expression of certain neuronal signaling and synaptic proteins. These findings provide new insights into how ovarian hormones and dietary composition affect the brain lipidome and amyloid burden. Furthermore, they strongly suggest that when designing dietary or pharmacological strategies to combat human neurodegenerative diseases, hormonal and metabolic status should be specifically taken into consideration as it may affect the therapeutic response.

SOD3 improves the tumor response to chemotherapy by stabilizing endothelial HIF-2α.

One drawback of chemotherapy is poor drug delivery to tumor cells, due in part to hyperpermeability of the tumor vasculature. Extracellular superoxide dismutase (SOD3) is an antioxidant enzyme usually repressed in the tumor milieu. Here we show that specific SOD3 re-expression in tumor-associated endothelial cells (ECs) increases doxorubicin (Doxo) delivery into and chemotherapeutic effect on tumors. Enhanced SOD3 activity fostered perivascular nitric oxide accumulation and reduced vessel leakage by inducing vascular endothelial cadherin (VEC) transcription. SOD3 reduced HIF prolyl hydroxylase domain protein activity, which increased hypoxia-inducible factor-2α (HIF-2α) stability and enhanced its binding to a specific VEC promoter region. EC-specific HIF-2α ablation prevented both the SOD3-mediated increase in VEC transcription and the enhanced Doxo effect. SOD3, VEC, and HIF-2α levels correlated positively in primary colorectal cancers, which suggests a similar interconnection of these proteins in human malignancy.

Dihydroceramide Desaturase 1 Inhibitors Reduce Amyloid-ß Levels in Primary Neurons from an Alzheimer's Disease Transgenic Model.

PURPOSE: The induction of autophagy has recently been explored as a promising therapeutic strategy to combat Alzheimer's disease. Among many other factors, there is evidence that ceramides/dihydroceramides act as mediators of autophagy, although the exact mechanisms underlying such effects are poorly understood. Here, we describe how two dihydroceramide desaturase inhibitors (XM461 and XM462) trigger autophagy and reduce amyloid secretion by neurons. METHODS: Neurons isolated from wild-type and APP/PS1 transgenic mice were exposed to the two dihydroceramide desaturase inhibitors to assess their effect on these cell's protein and lipid profiles. RESULTS: Both dihydroceramide desaturase inhibitors increased the autophagic vesicles in wild-type neurons, reflected as an increase in LC3-II, and this was correlated with the accumulation of dihydroceramides and dihydrosphingomyelins. Exposing APP/PS1 transgenic neurons to these inhibitors also produced a 50% reduction in amyloid secretion and/or production. The lipidomic defects triggered by these dihydroceramide desaturase inhibitors were correlated with a loss of S6K activity, witnessed by the changes in S6 phosphorylation, which strongly suggested a reduction of mTORC1 activity. CONCLUSIONS: The data obtained strongly suggest that dihydroceramide desaturase 1 activity may modulate autophagy and mTORC1 activity in neurons, inhibiting amyloid secretion and S6K activity. As such, it is tantalizing to propose that dihydroceramide desaturase 1 may be an important therapeutic target to combat amyloidosis.

Jaspine B induces nonapoptotic cell death in gastric cancer cells independently of its inhibition of ceramide synthase.

Sphingolipids (SLs) have been extensively investigated in biomedical research due to their role as bioactive molecules in cells. Here, we describe the effect of a SL analog, jaspine B (JB), a cyclic anhydrophytosphingosine found in marine sponges, on the gastric cancer cell line, HGC-27. JB induced alterations in the sphingolipidome, mainly the accumulation of dihydrosphingosine, sphingosine, and their phosphorylated forms due to inhibition of ceramide synthases. Moreover, JB provoked atypical cell death in HGC-27 cells, characterized by the formation of cytoplasmic vacuoles in a time and dose-dependent manner. Vacuoles appeared to originate from macropinocytosis and triggered cytoplasmic disruption. The pan-caspase inhibitor, z-VAD, did not alter either cytotoxicity or vacuole formation, suggesting that JB activates a caspase-independent cell death mechanism. The autophagy inhibitor, wortmannin, did not decrease JB-stimulated LC3-II accumulation. In addition, cell vacuolation induced by JB was characterized by single-membrane vacuoles, which are different from double-membrane autophagosomes. These findings suggest that JB-induced cell vacuolation is not related to autophagy and it is also independent of its action on SL metabolism.

Stereoselective preparation of quaternary 2-vinyl sphingosines and ceramides and their effect on basal sphingolipid metabolism.

The dicyclohexylborane-mediated addition of allene 1 to (E)-2-tridecenal affords a quaternary protected 2-amino-2-vinyl-1,3-diol in good yield as a single diastereomer. This compound is readily transformed into the four stereoisomers of the quaternary (E)-2-vinyl analogs of sphingosine. The metabolic fate and the effect of these compounds on the basal sphingolipid metabolism in human A549 lung adenocarcinoma cells has been studied, together with the ceramide analog of the most relevant vinylsphingosine derivative.

From the configurational preference of dihydroceramide desaturase-1 towards Δ6-unsaturated substrates to the discovery of a new inhibitor.

Dihydroceramide desaturase 1 (Des1) catalyzes the last step of ceramide synthesis de novo, thus regulating the physiologically relevant balance between dihydrosphingolipids and sphingolipids. Here we report on the configurational preference of Des1 towards isomeric Δ6-unsaturated dihydroceramide analogs and the discovery of a potent Des1 inhibitor.

Acid Ceramidase Deficiency in Mice Results in a Broad Range of Central Nervous System Abnormalities.

Farber disease is a rare autosomal recessive disorder caused by acid ceramidase deficiency that usually presents as early-onset progressive visceral and neurologic disease. To understand the neurologic abnormality, we investigated behavioral, biochemical, and cellular abnormalities in the central nervous system of Asah1P361R/P361R mice, which serve as a model of Farber disease. Behaviorally, the mutant mice had reduced voluntary locomotion and exploration, increased thigmotaxis, abnormal spectra of basic behavioral activities, impaired muscle grip strength, and defects in motor coordination. A few mutant mice developed hydrocephalus. Mass spectrometry revealed elevations of ceramides, hydroxy-ceramides, dihydroceramides, sphingosine, dihexosylceramides, and monosialodihexosylganglioside in the brain. The highest accumulation was in hydroxy-ceramides. Storage compound distribution was analyzed by mass spectrometry imaging and morphologic analyses and revealed involvement of a wide range of central nervous system cell types (eg, neurons, endothelial cells, and choroid plexus cells), most notably microglia and/or macrophages. Coalescing and mostly perivascular granuloma-like accumulations of storage-laden CD68+ microglia and/or macrophages were seen as early as 3 weeks of age and located preferentially in white matter, periventricular zones, and meninges. Neurodegeneration was also evident in specific cerebral areas in late disease. Overall, our central nervous system studies in Asah1P361R/P361R mice substantially extend the understanding of human Farber disease and suggest that this model can be used to advance therapeutic approaches for this currently untreatable disorder.

Mitochondrial GSH replenishment as a potential therapeutic approach for Niemann Pick type C disease.

Niemann Pick type C (NPC) disease is a progressive lysosomal storage disorder caused by mutations in genes encoding NPC1/NPC2 proteins, characterized by neurological defects, hepatosplenomegaly and premature death. While the primary biochemical feature of NPC disease is the intracellular accumulation of cholesterol and gangliosides, predominantly in endolysosomes, mitochondrial cholesterol accumulation has also been reported. As accumulation of cholesterol in mitochondria is known to impair the transport of GSH into mitochondria, resulting in mitochondrial GSH (mGSH) depletion, we investigated the impact of mGSH recovery in NPC disease. We show that GSH ethyl ester (GSH-EE), but not N-acetylcysteine (NAC), restored the mGSH pool in liver and brain of Npc1-/- mice and in fibroblasts from NPC patients, while both GSH-EE and NAC increased total GSH levels. GSH-EE but not NAC increased the median survival and maximal life span of Npc1-/- mice. Moreover, intraperitoneal therapy with GSH-EE protected against oxidative stress and oxidant-induced cell death, restored calbindin levels in cerebellar Purkinje cells and reversed locomotor impairment in Npc1-/- mice. High-resolution respirometry analyses revealed that GSH-EE improved oxidative phosphorylation, coupled respiration and maximal electron transfer in cerebellum of Npc1-/- mice. Lipidomic analyses showed that GSH-EE treatment had not effect in the profile of most sphingolipids in liver and brain, except for some particular species in brain of Npc1-/- mice. These findings indicate that the specific replenishment of mGSH may be a potential promising therapy for NPC disease, worth exploring alone or in combination with other options.

Dihydroceramide desaturase inhibitors induce autophagy via dihydroceramide-dependent and independent mechanisms.

BACKGROUND: Autophagy consists on the delivery of cytoplasmic material and organelles to lysosomes for degradation. Research on autophagy is a growing field because deciphering the basic mechanisms of autophagy is key to understanding its role in health and disease, and to paving the way to discovering novel therapeutic strategies. Studies with chemotherapeutic drugs and pharmacological tools support a role for dihydroceramides as mediators of autophagy. However, their effect on the autophagy outcome (cell survival or death) is more controversial. METHODS: We have examined the capacity of structurally varied Des1 inhibitors to stimulate autophagy (LC3-II analysis), to increase dihydroceramides (mass spectrometry) and to reduce cell viability (SRB) in T98G and U87MG glioblastoma cells under different experimental conditions. RESULTS: The compounds activity on autophagy induction took place concomitantly with accumulation of dihydroceramides, which occurred by both stimulation of ceramide synthesis de novo and reduction of Des1 activity. However, autophagy was also induced by the test compounds after preincubation with myriocin and in cells with a reduced capacity to produce dihydroceramides (U87DND). Autophagy inhibition with 3-methyladenine in the de novo dihydroceramide synthesis competent U87MG cells increased cytotoxicity, while genetic inhibition of autophagy in U87DND cells, poorly efficient at synthesizing dihydroceramides, augmented resistance to the test compounds. CONCLUSION: Dihydroceramide desaturase 1 inhibitors activate autophagy via both dihydroceramide-dependent and independent pathways and the balance between the two pathways influences the final cell fate. GENERAL SIGNIFICANCE: The cells capacity to biosynthesize dihydroceramides must be taken into account in proautophagic Des1 inhibitors-including therapies.

Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autolysosome destabilization.

Autophagy is considered primarily a cell survival process, although it can also lead to cell death. However, the factors that dictate the shift between these 2 opposite outcomes remain largely unknown. In this work, we used Δ9-tetrahydrocannabinol (THC, the main active component of marijuana, a compound that triggers autophagy-mediated cancer cell death) and nutrient deprivation (an autophagic stimulus that triggers cytoprotective autophagy) to investigate the precise molecular mechanisms responsible for the activation of cytotoxic autophagy in cancer cells. By using a wide array of experimental approaches we show that THC (but not nutrient deprivation) increases the dihydroceramide:ceramide ratio in the endoplasmic reticulum of glioma cells, and this alteration is directed to autophagosomes and autolysosomes to promote lysosomal membrane permeabilization, cathepsin release and the subsequent activation of apoptotic cell death. These findings pave the way to clarify the regulatory mechanisms that determine the selective activation of autophagy-mediated cancer cell death.