Glycosphingolipid Synthesis Research Articles

α-galactosidase A deficiency promotes von Willebrand factor secretion in models of Fabry disease

Fabry disease results from loss of activity of the lysosomal enzyme α-galactosidase A (GLA), leading to the accumulation of globoseries glycosphingolipids in vascular endothelial cells. Thrombosis and stroke are life-threatening complications of Fabry disease; however, the mechanism of the vasculopathy remains unclear. We explored the relationship between GLA deficiency and endothelial cell von Willebrand factor (VWF) secretion in invivo and invitro models of Fabry disease. Plasma VWF was significantly higher at two months and increased with age in Gla-null compared to wild-type mice. Disruption of GLA in a human endothelial cell line by siRNA and CRISPR/Cas9 resulted in a 3-fold and 5-fold increase in VWF secretion, respectively. The increase in VWF levels was associated with decreased endothelial nitric oxide synthase (eNOS) activity in both invitro models. Pharmacological approaches that increase nitric oxide bioavailability or decrease reactive oxygen species completely normalized the elevated VWF secretion in GLA deficient cells. In contrast, the abnormality was not readily reversed by recombinant human GLA or by inhibition of glycosphingolipid synthesis with eliglustat. These results suggest that GLA deficiency promotes VWF secretion through eNOS dysregulation, which may contribute to the vasculopathy of Fabry disease.

Inhibition of glycosphingolipid synthesis reverses skin inflammation and hair loss in ApoE\u2212/\u2212 mice fed western diet

Sphingolipids have been accorded numerous biological functions however, the effects of feeding a western diet (diet rich in cholesterol and fat) on skin phenotypes, and color is not known. Here, we observed that chronic high-fat and high-cholesterol diet intake in a mouse model of atherosclerosis (ApoE−/−) decreases the level of ceramides and glucosylceramide. At the expense of increased levels of lactosylceramide due to an increase in the expression of lactosylceramide synthase (GalT-V). This is accompanied with neutrophil infiltration into dermis, and enrichment of tumor necrosis factor-stimulated gene-6 (TSG-6) protein. This causes skin inflammation, hair discoloration and loss, in ApoE−/− mice. Conversely, inhibition of glycosphingolipid synthesis, by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), unbound or encapsulated in a biodegradable polymer (BPD) reversed these phenotypes. Thus, inhibition of glycosphingolipid synthesis represents a unique therapeutic approach relevant to human skin and hair Biology.

Synthetic Strategies for Modified Glycosphingolipids and Their Design as Probes.

The plasma membrane of cells contains a diverse array of lipids that provide important structural and biological features. Glycolipids are typically a minor component of the cell membrane and consist primarily of glycosphingolipids (GSLs). GSLs in vertebrates contain a multifarious assortment of glycan headgroups, which can be important to biological functions based on lipid-lipid and lipid-protein interactions. The design of probes to study these complex targets requires advanced synthetic methodologies. In this Review, we will discuss recent advances in chemical and chemoenzymatic synthesis of GSLs in conjunction with the use of these approaches to design new probes. Examples using either chemical or enzymatic semisynthesis methods starting from isolated GSLs will also be reviewed. Focusing primarily on vertebrate glycolipids, we will highlight examples of radionuclide, fluorophore, photoresponsive, and bioorthogonal tagged GSL probes.

ACBD3 is required for FAPP2 transferring glucosylceramide through maintaining the Golgi integrity.

Glycosphingolipid (GSL) metabolism is involved in various physiological processes, including all major cell signaling pathways, and its dysregulation is linked to some diseases. The four-phosphate adaptor protein FAPP2-mediated glucosylceramide (GlcCer) transport for complex GSL synthesis has been studied extensively. However, the molecular machinery of FAPP2 as a GlcCer-transferring protein remains poorly defined. Here, we identify a Golgi-resident protein, acyl-coenzyme A binding domain containing 3 (ACBD3), as an interacting partner of FAPP2. We find that ACBD3 knockdown leads to dramatic Golgi fragmentation, which subsequently causes FAPP2 dispersal throughout the cytoplasm and a decreased localization at trans-Golgi network. The further quantitative lipidomic analysis indicates that ACBD3 knockdown triggers abnormal sphingolipid metabolism. Interestingly, the expression of siRNA-resistant full-length ACBD3 can rescue these defects caused by ACBD3 knockdown. These data reveal critical roles for ACBD3 in maintaining the integrity of Golgi morphology and cellular sphingolipid homeostasis and establish the importance of the integrated Golgi complex for the transfer of GlcCer and complex GSL synthesis.

Recent neuroimaging, neurophysiological, and neuropathological advances for the understanding of NPC.

Niemann–Pick disease type C (NPC) is a rare autosomal recessive lysosomal storage disorder with extensive biological, molecular, and clinical heterogeneity. Recently, numerous studies have tried to shed light on the pathophysiology of the disease, highlighting possible disease pathways common to other neurodegenerative disorders, such as Alzheimer’s disease and frontotemporal dementia, and identifying possible candidate biomarkers for disease staging and response to treatment. Miglustat, which reversibly inhibits glycosphingolipid synthesis, has been licensed in the European Union and elsewhere for the treatment of NPC in both children and adults. A number of ongoing clinical trials might hold promise for the development of new treatments for NPC. The objective of the present work is to review and evaluate recent literature data in order to highlight the latest neuroimaging, neurophysiological, and neuropathological advances for the understanding of NPC pathophysiology. Furthermore, ongoing developments in disease-modifying treatments will be briefly discussed.

GM1 ganglioside-independent intoxication by Cholera toxin.

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.

Glycosphingolipids-function and useage.

Our research concerns the synthesis and function of glycosphingolipids (GSLs) in eukaryotic cells. Despite extensive structural characterization of these sugar-lipid conjugates, defining their function is difficult due to poor aqueous solubility. The fact that no one gene encodes their synthesis and membrane GSL organization within micro domains or ‘rafts’, is dependent on both their carbohydrate and lipid composition, further complicate functional studies. Membrane GSLs often provide the interface between pathogenic microbes and/or their products and host cells, making GSL-based therapeutics a possible mode of infection control. In turn, microbial GSL-binding ligands provide probes to study GSL function. The GSL lipid moiety and membrane environment regulate GSL receptor function and likely, biosynthesis. We have made species which mimic some of these features. Understanding the basis of this ‘aglycone’ effect is a central focus. We have found cholesterol masking of GSL carbohydrate via conformational restriction of the GSL carbohydrate is an important feature of GSL receptor function. A H-Bond network results in the carbohydrate adopting a membrane parallel rather than membrane perpendicular conformation. This renders the carbohydrate less available for trans-ligand binding-e.g. antibodies (invisible GSLs). The carbohydrate now forms an ‘umbrella’ over the steroid OH, restricting the access of cholesterol binding ligands also. Cholesterol masking of GSLs is particularly important in tumour cells. The higher tumour cell cholesterol content results in the masking of the majority of tumour associated GSLs from antibody recognition and thereby plays an important role in tumour evasion of immune surveillance. Strategies to deplete tumour cholesterol should reverse this masking. Several MAbs in clinical antineoplastic use, target GSLs and tumour biopsy binding is increased by cholesterol depletion. We have worked extensively on the E.coli elaborated AB5 verotoxin-VT (aka Shiga toxin-Stx) and showed the B subunit binds to the GSL, globotriaosyl ceramide (Gb3). Gb3 is upregulated in many human tumours ovarian, breast, renal, gastric, pancreatic, brain, colon, lymphoma, and their neovasculature. Thus VT has both anti-tumour and anti angiogenic potential. We showed VT efficacy in a brain tumour xenograft model and this has been verified with many other tumour models. The ABC cassette transporter and multiple drug resistance pump, MDR1 (and some homologues) plays an important role in the trans membrane supply of precursor GSL for the synthesis of Gb3 (and other GSLs). This means that MDR1 overexpression in drug resistant tumours can lead to increased Gb3 content and thereby increased VT sensitivity. Following ligand binding, cell surface GSLs undergo internalization and a process of retrograde transport from endosomes to the trans Golgi network to the ER. GSL binding bacterial toxins e.g. VT, cholera toxin use this pathway to target the ER where the A subunit is translocated to the cytosol via hijacking the ER retro translocon used for ER associated degradation (ERAD) of partially misfolded nascent proteins. We have used this property as a means to temporarily competitively block ERAD in the genetic diseases in which ERAD eliminates the mutant protein and induce/exacerbate disease symptoms (e.g. F508delta CFTR cystic fibrosis, Gaucher disease). This GSL dependent blockade partially reverses disease symptoms in cell culture and animal disease models, and provides a potential therapy for many misfolded protein based genetic diseases. ERAD blockade is also an antineoplastic stratagem. The HIV adhesion, gp120 also binds GSLs, including Gb3. We identified Gb3 as a host resistance factor against HIV and are using this to design novel inhibitors of HIV infection.

The Flow of Information from Nucleus to Golgi Is Contingent upon Nuclear Membrane Synthesis and Protraction of the Ceramide-Containing Membrane to Endoplasmic Reticulum

The nucleus-initiated augmentation of ER membrane is reflected in a coordinated synthesis and intercalation of the explicit proteins and lipids required for the replacement, repair and function of the cell and its organelles. The direct connection between nucleus and the membranes containing labeled sphingosine (SphN) and ceramide (Cer) was affirmed by determining synthetic activity of serine palmitoyltransferase (SPT). The SPT and the newly synthesized serine-labeled lipid products were identified in the Outer- and Inner-Nuclear Membrane (ONM, INM) and ER. The pulse-chase experiments disclosed that the incorporation of radiolabeled lipids into both nuclear membranes declined upon their simultaneous increase in Endoplasmic Reticulum (ER). These results, and prior findings regarding metabolic transfer of nuclear membrane phosphoinositides to the outer leaflet of ER [Slomiany and Slomiany, Health, 2011, 3, 187-199], allowed us to reason that INM and ONM are not distinct entities, but uninterrupted continuum facing nucleosol and then cytosol when protracted into segment known as ER. Consequently, the identification of SPT and its products in the inner leaflet of nuclear and ER microsomes lent credence to the luminal presence of Cer in Golgi, luminal synthesis of glycosphingolipids (GSphLs), sphingomyelin (SM), and their delivery to the outer leaflet of apical and basolateral cell membrane, respectively. The findings presented in this communication provide further support to our concept that the factual intercalation of proteins and lipids into the cell membranes can only take place during their simultaneous synthesis that is guided by the nuclear and cytosolic processes enacted in nuclear-ER membrane continuum. At the nuclear stage, the signal-specific genes expression promotes active synthesis and intercalation of lipids into the organelles’ customized membrane that is protracted and articulated in ER in form of transport vesicles.

Inhibition of hepatocellular carcinoma growth by blockade of glycosphingolipid synthesis.

Hepatocellular carcinoma (HCC) is one of the most frequent cancers. In vitro studies suggest that growth and response to therapy of human carcinomas may depend on glycosphingolipid (GSL) expression. Glucosylceramide synthase (GCS), encoded by the gene Ugcg, is the basic enzyme required for the synthesis of GSLs. Gene array analysis implied that Ugcg is significantly overexpressed in human HCC as compared to non-tumorous liver tissue. Therefore we have investigated whether tumor - genesis and - growth is altered in the absence of GSLs. An endogenous liver cancer model has been initiated by application of diethylnitrosamine in mice lacking Ugcg specifically in hepatocytes. We have now shown that hepatocellular tumor initiation and growth in mice is significantly inhibited by hepatic GSL deficiency in vivo. Neither the expression of cell cycle proteins, such as cyclins and pathways such as the MAP-kinase/Erk pathway nor the mTOR/Akt pathway as well as the number of liver infiltrating macrophages and T cells were essentially changed in tumors lacking GSLs. Significantly elevated bi-nucleation of atypical hepatocytes, a feature for impaired cytokinesis, was detected in tumors of mice lacking liver-specific GSLs. A reduction of proliferation and restricted growth of tumor microspheres due to delayed, GSL-dependent cytokinesis, analogous to the histopathologic phenotype in vivo could be demonstrated in vitro. GSL synthesis inhibition may thus constitute a potential therapeutic target for hepatocellular carcinoma.

Synthesis of glycosphingolipids from the fungus Hirsutella rhossiliensis

The total synthesis of two neutral glycosphingolipids (GSLs) from the fungus Hirsutella rhossiliensis has been achieved. The GSLs possess a common neogala-core (Galβ1-6Gal) and have the following sequence: α-d-Manp(1 → 3)-β-d-Galp(1 → 6)-β-d-Galp(1 → 6)-β-d-Galp(1↔1)Cer (1) and α-d-Manp(1 → 3)-β-d-Galp(1 → 6)[α-d-Glcp(1 → 4)]-β-d-Galp(1 → 6)-β-d-Galp(1↔1)Cer (2). Our efficient synthetic strategy uses the different reactivity of the hydroxyl groups of galactose and the α-orienting solvent effect of dioxane-toluene to generate GSLs (1) and (2).

WP1066, a small molecule inhibitor of the JAK/STAT3 pathway, inhibits ceramide glucosyltransferase activity

WP1066 is a well-known inhibitor of the JAK/STAT3 signaling pathway. By a screen of known small molecule inhibitors of various enzymes and protein factors, we identified WP1066 as a ceramide glucosyltransferase inhibitor. Ceramide glucosyltransferase catalyzes the first glycosylation step during glycosphingolipid synthesis. We found that WP1066 inhibited the activity of ceramide glucosyltransferase with an IC50 of 7.2 μM, and that its action was independent of JAK/STAT3 pathway blockade. Moreover, the modes of inhibition of ceramide glucosyltransferase were uncompetitive with respect to both C6-NBD-cermide and UDP-glucose.

Identification of a new B4GalNAcT1 (GM2/GD2/GA2 synthase) isoform, and regulation of enzyme stability and intracellular transport by arginine-based motif

Glycosphingolipids (GSLs) are abundant in plasma membranes of mammalian cells, and their synthesis is strictly regulated in the Golgi apparatus. Disruption of GSL homeostasis is the cause of numerous diseases. Hundreds of molecular species of GSLs exist, and the detailed mechanisms underlying their homeostasis remain unclear. We investigated the physiological significance of isoform production for β1,4-N-acetyl-galactosaminyl transferase 1/B4GALNT1 (B4GN1), an enzyme involved in synthesis of ganglio-series GSLs GM2/GD2/GA2. We discovered a new mRNA variant (termed variant 2) of B4GN1 through EST clone search. A new isoform, M1-B4GN1, which has an NH2-terminal cytoplasmic tail longer than that of previously-known isoform M2-B4GN1, is translated from variant 2. M1-B4GN1 has R-based motif (a retrograde transport signal) in the cytoplasmic tail. M1-B4GN1 is partially localized in the endoplasmic reticulum (ER) depending on the R-based motif, whereas M2-B4GN1 is localized in the Golgi. Stability of M1-B4GN1 is higher than that of M2-B4GN1 because of the R-based motif. M2-B4GN1 forms a homodimer via disulfide bonding. When M1-B4GN1 and M2-B4GN1 were co-expressed in CHO-K1 cells, the two isoforms formed a heterodimer. The M1/M2-B4GN1 heterodimer was more stable than the M2-B4GN1 homodimer, but the heterodimer was not transported from the Golgi to the ER. Our findings indicate that stabilization of M1-B4GN1 homodimer and M1/M2-B4GN1 heterodimer by R-based motif is related to prolongation of Golgi retention, but not to retrograde transport from the Golgi to the ER. Coexistence of several B4GN1 isoforms having distinctive characteristics presumably helps maintain overall enzyme stability and GSL homeostasis.

Incorporation of Fluorescence Ceramide-Based HPLC Assay for Rapidly and Efficiently Assessing Glucosylceramide Synthase In Vivo

Glucosylceramide synthase (GCS) is a rate-limiting enzyme catalyzing ceramide glycosylation, thereby regulating cellular ceramide levels and the synthesis of glycosphingolipids (GSLs) in cellular membranes. Alterations of GCS not only affect membrane integrity, but also closely correlate with stem cell pluripotency, cancer drug resistance, GSL storage disorders and other diseases. Enzyme activities measured conventionally with currently available ex-vivo methods do not enable reliable assessment of the roles played by GCS in vivo. We report herein a substrate-incorporation method enabling rapid and efficient assessment of GCS in-vivo activity. Upon nanoparticle-based delivery, fluorescent NBD C6-ceramide was efficiently converted to NBD C6-glucosylceramide in live cells or in mouse tissues, whereupon an HPLC assay enabled detection and quantification of NBD C6-glucosylceramide in the low-femtomolar range. The enzyme kinetics of GCS in live cells and mouse liver were well-described by the Michaelis-Menten model. GCS activities were significantly higher in drug-resistant cancer cells and in tumors overexpressing GCS, but reduced after silencing GCS expression or inhibiting this enzyme. Our studies indicate that this rapid and efficient method provides a valuable means for accurately assessing the roles played by GCS in normal vs. pathological states, including ones involving cancer drug resistance.

0656 SYMPTOMATIC NARCOLEPSY AMONG INHERITED DISORDER,SUCH AS NIEMANN-PICK TYPE C

The symptoms of narcolepsy can occur during the course of other neurological conditions. Inherited disorders, tumors and demyelinating diseases were the three most frequent causes for symptomatic narcolepsy. Among inherited disorders, Niemann-Pick type C (NPC), Myotonic dystrophy type 1 and Prader-Willi syndrome were major three disorders. NPC is an autosomal recessive and congenital neurological disorder characterized by the accumulation of cholesterol and glycosphingolipids in the peripheral tissues and in the brain. NPC had no effective treatment before, but since 2012, we can use Miglustat in Japan. Miglustat inhibits glucosylceramide synthase, an essential enzyme for the synthesis of most glycosphingolipids. In this study, 5 out of 10 cases have been treated with Miglustat. The 10 patients with NPC were included in the study. Patients or families were given informed consent for the lumbar puncture. We checked clinical symptoms, brain MRI, HLA and measured orexin levels. In this study, previous 5 cases were untreated by Miglustat, and recent 5 cases were treated by Miglustat. Six out of 10 cases had cataplexy. Four cases with normal orexin level (>200pg/ml) did not show cataplexy. The cases which orexin levels were low level (<110pg/ml) of intermediate levels (110-200pg/ml) exhibit cataplexy. In two cases with cataplexy, orexin levels at the onset were intermediate, and became lower in the later period. About recent one case, she had cataplexy with intermediate orexin level. Her cataplexy was reduced and orexin level was increased to normal level, and other symptom (dysphagia, and oculography) got better. The NPC patients with cataplexy have low or intermediate orexin levels. In the cases without cataplexy, their orexin levels were normal. Cataplexy and orexin measurement make a chance to early diagnose and treatment for NPC. a

Longitudinal study of the human gastrointestinal microbiota following fecal microbiota transplant (FMT) for Clostridium difficile infections

BackgroundClostridium difficile infection (CDI) is a nosocomial infection known to be the most crucial antecedent of healthcare‐associated diarrhea. Fecal microbiota transplant (FMT) has emerged as a successful treatment method when standard antibiotic therapy is ineffective for refractory CDI. However, our understanding is limited regarding the longitudinal differences in the gastrointestinal (GI) microbiota between patients with a successful or failed FMT.ObjectiveWe aimed to investigate the microbial structure and predictive metagenome functional content of FMT patients over a 90‐day period, and to determine if these outcomes differed in patients with a successful or failed procedure over time.MethodsPatient microbiome samples were assessed prior to FMT (n=8), and at 30 (n=4) and 90 days (n=4) post treatment. Treatment failure was defined as ≥3 episodes of diarrhea/day or, within 30 days, retreatment with antibiotics or FMT. Metagenome functional potential was predicted using PICRUSt (v. 1.0.0). PICRUSt data and relative abundances of microbial taxa were then analyzed using LEFSe (v. 1.0), a Linear Discriminant Analysis model which evaluated discriminant features using a Kruskall‐Wallis test for FMT status (success/fail) followed by Wilcoxon test for pairwise comparisons between timepoints (baseline/30‐d/90‐d).ResultsAcross timepoints, relative abundance of Fusobacteriaceae was significantly higher among patients with a failed FMT (p=0.015), and Enterococcaceae (p=0.020) and an unclassified family of Lactobacillales (p=0.023) were significantly greater in patients with a successful FMT. At the genus level, relative abundance of Lactococcus (p=0.027) and Fusobacterium (p=0.007) were higher in patients with failed FMT over time. Metagenome functional potential analyses revealed that enzymes related to steroid hormone and glycosphingolipid synthesis and glycosaminoglycan degradation were significantly higher in participants with a failed FMT over time.ConclusionsThe use of innovative bioinformatic tools in the present study permits identification of key differences in microbial community structures and functional gene potential between participants with a successful or failed FMT procedure across a 90‐day period. Differences in steroid hormone synthesis, glucocorticoid in particular, has been linked to increased mortality of CDI patients. An increase in the synthesis and metabolism of glycoconjugates have also been associated with an increase in the pathogenicity of microbes such as C. difficile. More research is required to determine if the gut microbial structure of patients with CDI can be used to predict if FMT is the best route for treatment.Support or Funding InformationPartial funding for this study was provided by the USDA National Institute of Food and Agriculture, Hatch project ILLU 538 384.

ORM: A Central Regulator of Sphingolipid Homeostasis and Composition in Arabidopsis

The sphingolipid metabolic network in Arabidopsis is tightly regulated to ensure sufficient biosynthetic flux to support cell viability and growth but to also rapidly respond to perturbations such as those induced by bacterial pathogenesis that trigger production of the bioactive sphingolipid metabolites. These metabolites include long‐chain bases and ceramides that signal programmed cell death (PCD) for plant defense. Regulation of sphingolipid biosynthesis is mediated primarily through reversible physical interactions, of serine palmitoyltransferase (SPT), the initial enzyme in long‐chain base biosynthesis, with ORM proteins (orosomucoid‐like proteins). Consistent with a role of ORMs as repressors of SPT activity, overexpression of the two Arabidopsis ORM proteins AtORM1 and AtORM2 strongly reduced the accumulation of free long‐chain bases in response to treatment with the mycotoxin fumonisin B1 (FB1), a ceramide synthase inhibitor. These plants also displayed increased resistance to the PCD‐inducing cytotoxicity of FB1. Conversely, RNAi suppression of AtORM1/2 resulted in large increases in long‐chain bases in response to FB1 treatment and increased sensitivity to FB1. AtORM1 and 2 overexpressing plants also had decreased activity of Class I ceramide synthase that generates ceramides with C16 fatty acids and dihydroxy long‐chain bases and increased activity of Class II ceramide synthases that produce ceramides with very‐long chain fatty acids and trihydroxy long‐chain bases. The reverse effect on ceramide synthase activities was observed in AtORM RNAi lines. These findings suggest an extended role of ORMs as regulators of the production of structurally and functionally distinct ceramides for glycosphingolipid synthesis to support plant growth and long‐chain base homeostasis.

Combination therapy in a patient with chronic neuronopathic Gaucher disease: a case report

BackgroundThe variants of neuronopathic Gaucher disease may be viewed as a clinical phenotypic continuum divided into acute and chronic forms. The chronic neuronopathic form of Gaucher disease is characterized by a later onset of neurological symptoms and protracted neurological and visceral involvement. The first-choice treatment for nonneuronopathic Gaucher disease is enzyme replacement therapy with recombinant analogues of the deficient human enzyme glucocerebrosidase. Enzyme replacement therapy has been shown to improve hematological and bone manifestations associated with Gaucher disease, but, as with most proteins, recombinant enzymes cannot cross the blood–brain barrier, which prevents effects on neurological manifestations. Substrate reduction therapy with miglustat (N-butyldeoxynojirimycin) inhibits glucosylceramide synthase, which catalyzes the first step in glycosphingolipid synthesis. Because miglustat can cross the blood–brain barrier, it has been suggested that, combined with enzyme replacement therapy, it might be effective in treating neurological symptoms in patients with neuronopathic Gaucher disease.Case presentationWe report observed effects of combined enzyme replacement therapy and substrate reduction therapy in a 7-year-old Caucasian boy with neuronopathic Gaucher disease who was homozygous for L444P mutations. He had received enzyme replacement therapy from the age of 18 months, and concomitant miglustat treatment was commenced, with dosing according to body surface area uptitrated over 1 month with dietary modifications when he reached the age of 30 months. He experienced mild diarrhea after commencing miglustat therapy, which decreased in frequency/severity over time. His splenomegaly was reduced, and his hematological values and plasma angiotensin-converting enzyme activity normalized. Plasma chitotriosidase also showed substantial and sustained decreases. After 5 years of combination therapy, the patient showed no signs of neurological impairment.ConclusionsThis case supports the concept that oral miglustat in combination with intravenous enzyme replacement therapy may be beneficial in preventing neurological signs in patients with chronic neuronopathic Gaucher disease. The importance of dietary modifications has also been confirmed. Further follow-up studies are needed to better define the therapeutic effect of combined treatment in this Gaucher disease subtype.

Lucerastat, an iminosugar with potential as substrate reduction therapy for glycolipid storage disorders: safety, tolerability, and pharmacokinetics in healthy subjects

BackgroundLucerastat, an inhibitor of glucosylceramide synthase, has the potential to restore the balance between synthesis and degradation of glycosphingolipids in glycolipid storage disorders such as Gaucher disease and Fabry disease. The safety, tolerability, and pharmacokinetics of oral lucerastat were evaluated in two separate randomized, double-blind, placebo-controlled, single- and multiple-ascending dose studies (SAD and MAD, respectively) in healthy male subjects.MethodsIn the SAD study, 31 subjects received placebo or a single oral dose of 100, 300, 500, or 1000 mg lucerastat. Eight additional subjects received two doses of 1000 mg lucerastat or placebo separated by 12 h. In the MAD study, 37 subjects received placebo or 200, 500, or 1000 mg b.i.d. lucerastat for 7 consecutive days. Six subjects in the 500 mg cohort received lucerastat in both absence and presence of food.ResultsIn the SAD study, 15 adverse events (AEs) were reported in ten subjects. Eighteen AEs were reported in 15 subjects in the MAD study, in which the 500 mg dose cohort was repeated because of elevated alanine aminotransferase (ALT) values in 4 subjects, not observed in other dose cohorts. No severe or serious AE was observed. No clinically relevant abnormalities regarding vital signs and 12–lead electrocardiograms were observed. Lucerastat Cmax values were comparable between studies, with geometric mean Cmax 10.5 (95% CI: 7.5, 14.7) and 11.1 (95% CI: 8.7, 14.2) μg/mL in the SAD and MAD study, respectively, after 1000 mg lucerastat b.i.d. tmax (0.5 – 4 h) and t1/2 (3.6 – 8.1 h) were also within the same range across dose groups in both studies. Using the Gough power model, dose proportionality was confirmed in the SAD study for Cmax and AUC0–∞, and for AUC0–12 in the MAD study. Fed-to-fasted geometric mean ratio for AUC0–12 was 0.93 (90% CI: 0.80, 1.07) and tmax was the same with or without food, indicating no food effect.ConclusionsIncidence of drug-related AEs did not increase with dose. No serious AEs were reported for any subject. Overall, lucerastat was well tolerated. These results warrant further investigation of substrate reduction therapy with lucerastat in patients with glycolipid storage disorders.SAD study was registered on clinicaltrials.gov under the identifier NCT02944487 on the 24th of October 2016 (retrospectively registered). MAD study was registered on clinicaltrials.gov under the identifier NCT02944474 on the 25th of October 2016 (retrospectively registered).Trial registrationA Study to Assess the Safety and Tolerability of Lucerastat in Subjects With Fabry Disease. Clinicaltrials.gov: NCT02930655.

PDMP, a ceramide analogue, acts as an inhibitor of mTORC1 by inducing its translocation from lysosome to endoplasmic reticulum

Mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism, and cell differentiation. Recent studies have revealed that the recruitment of mTORC1 to lysosomes is essential for its activation. The ceramide analogue 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a well known glycosphingolipid synthesis inhibitor, also affects the structures and functions of various organelles, including lysosomes and endoplasmic reticulum (ER). We investigated whether PDMP regulates the mTORC1 activity through its effects on organellar behavior. PDMP induced the translocation of mTORC1 from late endosomes/lysosomes, leading to the dissociation of mTORC1 from its activator Rheb in MC3T3-E1 cells. Surprisingly, we found mTORC1 translocation to the ER upon PDMP treatment. This effect of PDMP was independent of its action as the inhibitor, since two stereoisomers of PDMP, with and without the inhibitor activity, showed essentially the same effect. We confirmed that PDMP inhibits the mTORC1 activity based on the decrease in the phosphorylation of ribosomal S6 kinase, a downstream target of mTORC1, and the increase in LC3 puncta, reflecting autophagosome formation. Furthermore, PDMP inhibited the mTORC1-dependent osteoblastic cell proliferation and differentiation of MC3T3-E1 cells. Accordingly, the present results reveal a novel mechanism of PDMP, which inhibits the mTORC1 activity by inducing the translocation of mTOR from lysosomes to the ER.

Glycosphingolipid-Protein Interaction in Signal Transduction.

Glycosphingolipids (GSLs) are a class of ceramide-based glycolipids essential for embryo development in mammals. The synthesis of specific GSLs depends on the expression of distinctive sets of GSL synthesizing enzymes that is tightly regulated during development. Several reports have described how cell surface receptors can be kept in a resting state or activate alternative signalling events as a consequence of their interaction with GSLs. Specific GSLs, indeed, interface with specific protein domains that are found in signalling molecules and which act as GSL sensors to modify signalling responses. The regulation exerted by GSLs on signal transduction is orthogonal to the ligand–receptor axis, as it usually does not directly interfere with the ligand binding to receptors. Due to their properties of adjustable production and orthogonal action on receptors, GSLs add a new dimension to the control of the signalling in development. GSLs can, indeed, dynamically influence progenitor cell response to morphogenetic stimuli, resulting in alternative differentiation fates. Here, we review the available literature on GSL–protein interactions and their effects on cell signalling and development.