Inhibition Of Glycosphingolipid Synthesis Research Articles

A case report of Tangier disease presents with acute sensorimotor polyneuropathy and its treatment approach

Polyneuropathy is a frequently encountered clinical presentation where peripheral nerves are affected due to the same cause and physiopathological processes. We report a case of acute sensorimotor polyneuropathy in a patient with Tangier disease (TD) who was treated with miglustat which is a glycosphingolipid synthesis inhibitor. TD is a very rare genetic disorder caused by mutations in the ATP-binding cassette transporter A1 (ABCA1) gene which encodes the cholesterol efflux regulatory protein. It leads to accumulation of cholesterol esters within various tissues and affects lipid metabolism by deficiency of high-density lipoprotein (HDL) in the blood. Due to the accumulation of cholesterol esters in Schwann cells, it could provoke polyneuropathy in TD. Our case presented to our clinic with quadriparesis and after treated with miglustat therapy his weakness regressed.

A differential proteomics study of cerebrospinal fluid from individuals with Niemann-Pick disease, Type C1.

Niemann-Pick, type C1 (NPC1) is a fatal, neurodegenerative disease, which belongs to the family of lysosomal diseases. In NPC1, endo/lysosomal accumulation of unesterified cholesterol and sphingolipids arise from improper intracellular trafficking resulting in multi-organ dysfunction. With the proximity between the brain and cerebrospinal fluid (CSF), performing differential proteomics provides a means to shed light to changes occurring in the brain. In this study, CSF samples obtained from NPC1 individuals and unaffected controls were used for protein biomarker identification. A subset of these individuals with NPC1 are being treated with miglustat, a glycosphingolipid synthesis inhibitor. Of the 300 identified proteins, 71 proteins were altered in individuals with NPC1 compared to controls including cathepsin D, and members of the complement family. Included are a report of 10 potential markers for monitoring therapeutic treatment. We observed that pro-neuropeptide Y (NPY) was significantly increased in NPC1 individuals relative to healthy controls; however, individuals treated with miglustat displayed levels comparable to healthy controls. In further investigation, NPY levels in a NPC1 mouse model corroborated our findings. We posit that NPY could be a potential therapeutic target for NPC1 due to its multiple roles in the central nervous system such as attenuating neuroinflammation and reducing excitotoxicity.

Host lactosylceramide enhances Edwardsiella tarda infection.

Edwardsiella tarda is a Gram-negative bacterium causing economic damage in aquaculture. The interaction of E. tarda with microdomains is an important step in the invasion, but the target molecules in microdomains remain undefined. Here, we found that intraperitoneal injection of E. tarda altered splenic glycosphingolipid patterns in the model host medaka (Oryzias latipes) accompanied by alteration of glycosphingolipid metabolism-related gene expressions, suggesting that glycosphingolipid levels are involved in E. tarda infection. To ascertain the significance of glycosphingolipids in the infection, fish cell lines, DIT29 cells with a high amount of lactosylceramide (LacCer) and glucosylceramide (GlcCer), and GAKS cells with a low amount of these lipids, were treated with methyl-β-cyclodextrin to disrupt the microdomain. E. tarda infection was suppressed in DIT29 cells, but not in GAKS cells, suggesting the involvement of microdomain LacCer and GlcCer in the infection. DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, an inhibitor of glycosphingolipid-synthesis, attenuated the infection in DIT29 cells, while Neu3-overexpressing GAKS cells, which accumulated LacCer, enhanced the infection. E. tarda possessed binding ability towards LacCer, but not GlcCer, and LacCer preincubation declined the infection towards fish cells, possibly due to the masking of binding sites. The present study suggests that LacCer may be a positive regulator of E. tarda invasion.

The SPPL3-Defined Glycosphingolipid Repertoire Orchestrates HLA Class I-Mediated Immune Responses

HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ Tcells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ Tcell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.

P.230 Treatment-resistant psychotic symptoms in non-syndromic intellectual disability involving an interstitial deletion of 20q13.13

Adult onset Niemann- Pick disease type C (NPC) is a rare autosomal recessive lysosomal storage disorder that presents with heterogenous neuropsychiatric symptoms. Because of the variation of presentations, the diagnosis is often delayed or overlooked. Another treatment challenge is that currently there is only one FDA approved treatment, Miglustat, a reversible glycosphingolipid synthesis inhibitor, and it takes up to six months for therapeutic effect.We present a case report of a 27 year-old woman who was admitted to a medical hospital for inpatient work-up of altered mental status. Her initial symptoms were mild impairments in attention, bradykinesia, and ataxia that eventually progressed to an episode of acute encephalopathy with psychosis and catatonia. She underwent a comprehensive neurologic and medical work-up which was non-revealing, and then had genetic testing which revealed NPC. She was trialed on multiple medications in an attempt to manage her neuropsychiatric symptoms with minimal benefit. The decision was made to proceed with electroconvulsive therapy (ECT) with marked improvement in her symptoms after just one treatment, and continued response throughout all 18 treatments she has received thus far. While the patient has had relapses in her encephalopathic symptoms during the course of ECT treatments, additional ECT treatments following have been the most effective in returning her to her new baseline and allowing her to remain functional during this time.

Correction of cilia structure and function alleviates multi-organ pathology in Bardet-Biedl syndrome mice.

Bardet–Biedl syndrome (BBS) is a pleiotropic autosomal recessive ciliopathy affecting multiple organs. The development of potential disease-modifying therapy for BBS will require concurrent targeting of multi-systemic manifestations. Here, we show for the first time that monosialodihexosylganglioside accumulates in Bbs2−/− cilia, indicating impairment of glycosphingolipid (GSL) metabolism in BBS. Consequently, we tested whether BBS pathology in Bbs2−/− mice can be reversed by targeting the underlying ciliary defect via reduction of GSL metabolism. Inhibition of GSL synthesis with the glucosylceramide synthase inhibitor Genz-667161 decreases the obesity, liver disease, retinal degeneration and olfaction defect in Bbs2−/− mice. These effects are secondary to preservation of ciliary structure and signaling, and stimulation of cellular differentiation. In conclusion, reduction of GSL metabolism resolves the multi-organ pathology of Bbs2−/− mice by directly preserving ciliary structure and function towards a normal phenotype. Since this approach does not rely on the correction of the underlying genetic mutation, it might translate successfully as a treatment for other ciliopathies.

The SPPL3-Defined Glycosphingolipid Repertoire Regulates Immune Responses by Improving HLA Class I Access

HLA class I (HLA-I) drives immune responses by presenting antigen-derived peptides to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Since therapeutic options for restoring HLA-I antigen presentation are limited, we aimed to identify new HLA-I pathway targets. By iterative genome-wide screens we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augments B3GNT5 enzyme activity, resulting in upregulated levels of surface (neo)lacto-series GSLs. These GSLs sterically impede molecular interactions with HLA-I and diminish CD8+ T cell activation. In accordance, a disturbed SPPL3-B3GNT5 pathway in glioma associates with decreased patient survival. Importantly, we show that this immunomodulatory effect can be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that functionally inhibits antigen presentation and represents a potential therapeutic target in cancer, infection and autoimmunity.

Management of metabolic syndrome and reduction in body weight in type II diabetic mice by inhibiting glycosphingolipid synthesis

Metabolic syndrome is defined by hyperlipidemia and cardiovascular complications. We have examined whether inhibition of glycosphingolipid synthesis can interfere with metabolic syndrome in a male mouse model of type II diabetes (db/db). The db/db and control mice (C57/BL6) (n = 6) fed chow for 30 weeks received vehicle (5% Tween-80 in PBS; 100 μl), or a biopolymer-encapsulated D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (BPD) glycosphingolipid synthesis inhibitor daily via oral gavage for 6 weeks. Echocardiography revealed increased Ao-IMT in db/db mice compared to control. However, BPD decreased Ao-IMT, monohexosylceramide and dihexosylceramide, LDL, triglycerides, glucose, and raised HDL levels in db/db mice. This was due to increased gene expression of HMG-CoA reductase, LDLr, SREBP2, and bile acids: Cy7-a hydroxylase, LXR and FXR, lipoprotein lipase, VLDL receptor and PPAR. Treatment also increased the expression of superoxide dismutase-II to reduce the pro-oxidant status in these mice. We observed that decreased cholesterol levels correlated with decreased cholesterol sensing proteins e.g. NPC1 gene/protein expression and mammalian target of rapamycin (mTORC-1) and reduced body weight. Thus, glycosphingolipid synthesis inhibition is a novel approach to manage metabolic syndrome and reduce body weight in diabetic mice and with potential applications in humans.

Lactosylceramide synthase β-1,4-GalT-V: A novel target for the diagnosis and therapy of human colorectal cancer

Little is known about an oncogenic signal transducer β-1,4-galactosyltransferase-V (β-1,4-GalT-V), in human colorectal cancer. Using quantitative RT-PCR, immunohistochemical staining and ELISA assays, we determined that β-1,4-GalT-V gene/protein expression is specifically increased in human colorectal cancer (CRC) tumors, compared to visibly normal tissue. Furthermore, we observed a marked increase in its enzymatic activity, and its product lactosylceramide. Moreover, we found increased dihydrosphingolipid metabolites, in particular dihydrosphingomyelin in cancer tissue compared to normal. Further, inhibition of glycosphingolipid synthesis by the synthetic ceramide analog, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), concurrently inhibited colorectal cancer cell (HCT-116) proliferation, as well as β-1,4-GalT-V mass and several glycosphingolipid levels. We conclude that β-1,4-GalT-V may serve as a diagnostic and therapeutic biomarker for the progression of human colorectal cancer, and consequently, inhibition of GSL synthesis may be a novel approach for the treatment of this life-threatening disease.

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.

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.

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.

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.

Anti-amyloidogenic effects of glycosphingolipid synthesis inhibitors occur independently of ganglioside alterations.

Evidence has suggested that ganglioside abnormalities may be linked to the proteolytic processing of amyloid precursor protein (APP) in Alzheimer's disease (AD) and that pharmacological inhibition of ganglioside synthesis may reduce amyloid β-peptide (Aβ) production. In this study, we assessed the usefulness of two well-established glycosphingolipid (GSL) synthesis inhibitors, the synthetic ceramide analog D-PDMP (1-phenyl 2-decanoylamino-3-morpholino-1-propanol) and the iminosugar N-butyldeoxynojirimycin (NB-DNJ or miglustat), as anti-amyloidogenic drugs in a human cellular model of AD. We found that both GSL inhibitors were able to markedly inhibit Aβ production, although affecting differently the APP cleavage. Surprisingly, the L-enantiomer of PDMP, which promotes ganglioside accumulation, acted similarly to D-PDMP to inhibit Aβ production. Concurrently, both D- and L-PDMP strongly and equally reduced the levels of long-chain ceramides. Altogether, our data suggested that the anti-amyloidogenic effects of PDMP agents are independent of the altered cellular ganglioside composition, but may result, at least in part, from their ability to reduce ceramide levels. Moreover, our current study established for the first time that NB-DNJ, a drug already used as a therapeutic for Gaucher disease (a lysosomal storage disorder), was also able to reduce Aβ production in our cellular model. Therefore, our study provides novel information regarding the possibilities to target amyloidogenic processing of APP through modulation of sphingolipid metabolism and emphasizes the potential of the iminosugar NB-DNJ as a disease modifying therapy for AD.

Biochemical response to substrate reduction therapy versus enzyme replacement therapy in Gaucher disease type 1 patients.

BackgroundWe retrospectively compared biochemical responses in type 1 Gaucher disease patients to treatment with glycosphingolipid synthesis inhibitors miglustat and eliglustat and ERT.MethodsSeventeen GD1 patients were included (n = 6 eliglustat, (two switched from ERT), n = 9 miglustat (seven switchers), n = 4 ERT (median dose 60U/kg/m). Plasma protein markers reflecting disease burden (chitotriosidase, CCL18) and lipids reflecting substrate accumulation (glucosylsphingosine, glucosylceramide) were determined. Also, liver and spleen volumes, hemoglobin, platelets, and fat fraction were measured.ResultsIn patients naïve to treatment, chitotriosidase, CCL18 and glucosylsphingosine decreased comparably upon eliglustat and ERT treatment, while the response to miglustat was less. After 2 years, median decrease of chitotriosidase was 89 % (range 77–98), 88 % (78–92) and 37 % (29–46) for eliglustat, ERT and miglustat naïve patients respectively; decrease of CCL18 was 73 % (63–78), 54 % (43–86), and 10 % (3–18); decrease of glucosylsphingosine was 86 % (78–93), 78 % (65–91), 48 % (46–50). Plasma glucosylceramide in eliglustat treated patients (n = 4) reached values below the normal range (n = 20 healthy controls). Biochemical markers decreased or stabilized in switchers from ERT to eliglustat (n = 2), but less in miglustat switchers (n = 7). Clinical parameters responded comparably upon eliglustat and ERT treatment.ConclusionsOur explorative study provides evidence that biochemical markers respond comparably in patients receiving eliglustat treatment and ERT, while the corresponding response to miglustat treatment is less.

Normalisation of brain spectroscopy findings in Niemann-Pick disease type C patients treated with miglustat.

Niemann–Pick disease type C (NP-C) is a fatal progressive neurolipidosis involving neuronal storage of cholesterol and gangliosides. Miglustat, an inhibitor of glycosphingolipid synthesis, has been approved to treat neurological manifestations in adults and children with NP-C. This open-label observational study in adults with confirmed NP-C evaluated the efficacy of miglustat (200 mg t.i.d.) based on composite functional disability (CFD) scores and brain proton magnetic resonance spectroscopy (H-MRS) measurement of choline (Cho)/N-acetyl aspartate (NAA) ratio in the centrum ovale. Overall, 16 patients were included and received miglustat for a mean period of 30.6 months: 12 continued on miglustat throughout follow up, and 4 discontinued miglustat because of adverse effects (n = 2) or perceived lack of efficacy (n = 2). In the ‘continued’ subgroup, the mean (SD) annual progression of CFD scores decreased from 0.75 (0.94) before treatment to 0.29 (1.29) during the period between miglustat initiation and last follow-up. In the discontinued subgroup, CFD progression increased from 0.48 (0.44) pre-treatment to 1.49 (1.31) at last follow up (off treatment). Mean (SD) Cho/NAA ratio [normal level 0.48 (0.076)] decreased during miglustat treatment in the continued subgroup: 0.64 (0.12) at baseline (miglustat initiation), 0.59 (0.17) at 12-month follow up, and 0.48 (0.09) at 24-month follow up. Cho/NAA ratio remained relatively stable in the discontinued subgroup: 0.57 (0.15), 0.53 (0.04) and 0.55 (0.09), respectively. In conclusion, H-MRS Cho/NAA ratio might serve as an objective, quantitative neurological marker of brain dysfunction in NP-C, allowing longitudinal analysis of the therapeutic effect of miglustat.Electronic supplementary materialThe online version of this article (doi:10.1007/s00415-016-8051-1) contains supplementary material, which is available to authorized users.

Gpnmb Is a Potential Marker for the Visceral Pathology in Niemann-Pick Type C Disease.

Impaired function of NPC1 or NPC2 lysosomal proteins leads to the intracellular accumulation of unesterified cholesterol, the primary defect underlying Niemann-Pick type C (NPC) disease. In addition, glycosphingolipids (GSLs) accumulate in lysosomes as well. Intralysosomal lipid accumulation triggers the activation of a set of genes, including potential biomarkers. Transcript levels of Gpnmb have been shown to be elevated in various tissues of an NPC mouse model. We speculated that Gpnmb could serve as a marker for visceral lipid accumulation in NPC disease. We report that Gpnmb expression is increased at protein level in macrophages in the viscera of Npc1nih/nih mice. Interestingly, soluble Gpnmb was also found to be increased in murine and NPC patient plasma. Exposure of RAW264.7 macrophages to the NPC-phenotype-inducing drug U18666A also upregulated Gpnmb expression. Inhibition of GSL synthesis with the glucosylceramide synthase (GCS) inhibitor N-butyl-1-deoxynojirimycin prevented U18666A-induced Gpnmb induction and secretion. In summary, we show that Gpnmb is upregulated in NPC mice and patients, most likely due to GSL accumulation.

Prevention of cardiac hypertrophy by the use of a glycosphingolipid synthesis inhibitor in ApoE−/− mice

ApoE−/− mice fed a high fat and high cholesterol (HFHC) diet (20% fat and 1.25% cholesterol) from 12 weeks of age to 36 weeks revealed an age-dependent increase in the left ventricular mass (LV mass) and decline in fractional shortening (FS%), which worsened with HFHC diet. These traits are indicative of maladaptive pathological cardiac hypertrophy and dysfunction. This was accompanied by loading of glycosphingolipids and increased gene expression of ANP, BNP in myocardial tissue. Masson's trichrome staining revealed a significant increase in cardiomyocyte size and fibrosis. In contrast, treatment with 5 and 10 μM D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of glucosylceramide synthase and lactosylceramide synthase, dose-dependently decreased the load of glycosphingolipids and preserved fractional shortening and maintained left ventricular mass to normal 12-week-old control levels over a 6 month treatment period. Our mechanistic studies showed that D-PDMP inhibited cardiac hypertrophy by inhibiting the phosphorylation of mitogen–activated protein kinase (MAPK). We propose that associating increased glycosphingolipid synthesis with cardiac hypertrophy could serve as a novel approach to prevent this phenotype in experimental animal models of diet -induced atherosclerotic heart disease.

Glycosphingolipids are modulators of disease pathogenesis in amyotrophic lateral sclerosis

Recent genetic evidence suggests that aberrant glycosphingolipid metabolism plays an important role in several neuromuscular diseases including hereditary spastic paraplegia, hereditary sensory neuropathy type 1, and non-5q spinal muscular atrophy. Here, we investigated whether altered glycosphingolipid metabolism is a modulator of disease course in amyotrophic lateral sclerosis (ALS). Levels of ceramide, glucosylceramide, galactocerebroside, lactosylceramide, globotriaosylceramide, and the gangliosides GM3 and GM1 were significantly elevated in spinal cords of ALS patients. Moreover, enzyme activities (glucocerebrosidase-1, glucocerebrosidase-2, hexosaminidase, galactosylceramidase, α-galactosidase, and β-galactosidase) mediating glycosphingolipid hydrolysis were also elevated up to threefold. Increased ceramide, glucosylceramide, GM3, and hexosaminidase activity were also found in SOD1(G93A) mice, a familial model of ALS. Inhibition of glucosylceramide synthesis accelerated disease course in SOD1(G93A) mice, whereas infusion of exogenous GM3 significantly slowed the onset of paralysis and increased survival. Our results suggest that glycosphingolipids are likely important participants in pathogenesis of ALS and merit further analysis as potential drug targets.

Improved intervention of atherosclerosis and cardiac hypertrophy through biodegradable polymer-encapsulated delivery of glycosphingolipid inhibitor

D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a glycosphingolipid synthesis inhibitor, holds promise for the treatment of atherosclerosis and cardiac hypertrophy but rapid in vivo clearance has severely hindered translation to the clinic. To overcome this impediment, we used a materials-based delivery strategy wherein D-PDMP was encapsulated within a biodegradable polymer composed of poly ethylene glycol (PEG) and sebacic acid (SA). PEG-SA was formulated into nanoparticles that were doped with 125I-labeled PEG to allow in vivo bio-distribution and release kinetics of D-PDMP to be determined by using γ-scintigraphy and subsequently, by mass spectrometry. Polymer-encapsulation increased the residence time of D-PDMP in the body of a treated mouse from less than one hour to at least four hours (and up to 48 h or longer). This substantially increased in vivo longevity provided by polymer encapsulation resulted in an order of magnitude gain in efficacy for interfering with atherosclerosis and cardiac hypertrophy in apoE−/− mice fed a high fat and high cholesterol (HFHC) diet. These results establish that D-PDMP encapsulated in a biodegradable polymer provides a superior mode of delivery compared to unconjugated D-PDMP by way of increased gastrointestinal absorption and increased residence time thus providing this otherwise rapidly cleared compound with therapeutic relevance in interfering with atherosclerosis, cardiac hypertrophy, and probably other diseases associated with the deleterious effects of abnormally high glycosphingolipid biosynthesis or deficient catabolism.