GlcCer Accumulation Research Articles

Glucosylceramide in bunyavirus particles is essential for virus binding to host cells

Hexosylceramides (HexCer) are implicated in the infection process of various pathogens. However, the molecular and cellular functions of HexCer in infectious cycles are poorly understood. Investigating the enveloped virus Uukuniemi (UUKV), a bunyavirus of the Phenuiviridae family, we performed a lipidomic analysis with mass spectrometry and determined the lipidome of both infected cells and derived virions. We found that UUKV alters the processing of HexCer to glycosphingolipids (GSL) in infected cells. The infection resulted in the overexpression of glucosylceramide (GlcCer) synthase (UGCG) and the specific accumulation of GlcCer and its subsequent incorporation into viral progeny. UUKV and several pathogenic bunyaviruses relied on GlcCer in the viral envelope for binding to various host cell types. Overall, our results indicate that GlcCer is a structural determinant of virions crucial for bunyavirus infectivity. This study also highlights the importance of glycolipids on virions in facilitating interactions with host cell receptors and infectious entry of enveloped viruses.

Glucosylceramide flippases contribute to cellular glucosylceramide homeostasis

Lipid transport is an essential cellular process with importance to human health, disease development, and therapeutic strategies. Type IV P-type ATPases (P4-ATPases) have been identified as membrane lipid flippases by utilizing nitrobenzoxadiazole (NBD)-labeled lipids as substrates. Among the 14 human type IV P-type ATPases, ATP10D was shown to flip NBD-glucosylceramide (GlcCer) across the plasma membrane. Here, we found that conversion of incorporated GlcCer (d18:1/12:0) to other sphingolipids is accelerated in cells exogenously expressing ATP10D but not its ATPase-deficient mutant. These findings suggest that 1) ATP10D flips unmodified GlcCer as well as NBD-GlcCer at the plasma membrane and 2) ATP10D can translocate extracellular GlcCer, which is subsequently converted to other metabolites. Notably, exogenous expression of ATP10D led to the reduction in cellular hexosylceramide levels. Moreover, the expression of GlcCer flippases, including ATP10D, also reduced cellular hexosylceramide levels in fibroblasts derived from patients with Gaucher disease, which is a lysosomal storage disorder with excess GlcCer accumulation. Our study highlights the contribution of ATP10D to the regulation of cellular GlcCer levels and maintaining lipid homeostasis.

Identification of GM1-Ganglioside Secondary Accumulation in Fibroblasts from Neuropathic Gaucher Patients and Effect of a Trivalent Trihydroxypiperidine Iminosugar Compound on Its Storage Reduction.

Gaucher disease (GD) is a rare genetic metabolic disorder characterized by a dysfunction of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) due to mutations in the gene GBA1, leading to the cellular accumulation of glucosylceramide (GlcCer). While most of the current research focuses on the primary accumulated material, lesser attention has been paid to secondary storage materials and their reciprocal intertwining. By using a novel approach based on flow cytometry and fluorescent labelling, we monitored changes in storage materials directly in fibroblasts derived from GD patients carrying N370S/RecNcil and homozygous L444P or R131C mutations with respect to wild type. In L444P and R131C fibroblasts, we detected not only the primary accumulation of GlcCer accumulation but also a considerable secondary increase in GM1 storage, comparable with the one observed in infantile patients affected by GM1 gangliosidosis. In addition, the ability of a trivalent trihydroxypiperidine iminosugar compound (CV82), which previously showed good pharmacological chaperone activity on GCase enzyme, to reduce the levels of storage materials in L444P and R131C fibroblasts was tested. Interestingly, treatment with different concentrations of CV82 led to a significant reduction in GM1 accumulation only in L444P fibroblasts, without significantly affecting GlcCer levels. The compound CV82 was selective against the GCase enzyme with respect to the β-Galactosidase enzyme, which was responsible for the catabolism of GM1 ganglioside. The reduction in GM1-ganglioside level cannot be therefore ascribed to a direct action of CV82 on β-Galactosidase enzyme, suggesting that GM1 decrease is rather related to other unknown mechanisms that follow the direct action of CV82 on GCase. In conclusion, this work indicates that the tracking of secondary storages can represent a key step for a better understanding of the pathways involved in the severity of GD, also underlying the importance of developing drugs able to reduce both primary and secondary storage-material accumulations in GD.

Axonal Transport of Lysosomes Is Unaffected in Glucocerebrosidase-Inhibited iPSC-Derived Forebrain Neurons.

Lysosomes are acidic organelles that traffic throughout neurons delivering catabolic enzymes to distal regions of the cell and maintaining degradative demands. Loss of function mutations in the gene GBA encoding the lysosomal enzyme glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher's disease (GD) and are the most common genetic risk factor for synucleinopathies like Parkinson's disease (PD) and dementia with Lewy bodies (DLB). GCase degrades the membrane lipid glucosylceramide (GlcCer) and mutations in GBA, or inhibiting its activity, results in the accumulation of GlcCer and disturbs the composition of the lysosomal membrane. The lysosomal membrane serves as the platform to which intracellular trafficking complexes are recruited and activated. Here, we investigated whether lysosomal trafficking in axons was altered by inhibition of GCase with the pharmacological agent Conduritol B Epoxide (CBE). Using live cell imaging in human male induced pluripotent human stem cell (iPSC)-derived forebrain neurons, we demonstrated that lysosomal transport was similar in both control and CBE-treated neurons. Furthermore, we tested whether lysosomal rupture, a process implicated in various neurodegenerative disorders, was affected by inhibition of GCase. Using L-leucyl-L-leucine methyl ester (LLoME) to induce lysosomal membrane damage and immunocytochemical staining for markers of lysosomal rupture, we found no difference in susceptibility to rupture between control and CBE-treated neurons. These results suggest the loss of GCase activity does not contribute to neurodegenerative disease by disrupting either lysosomal transport or rupture.

Consequences of excessive glucosylsphingosine in glucocerebrosidase-deficient zebrafish.

AbstractIn Gaucher disease (GD), the deficiency of glucocerebrosidase causes lysosomal accumulation of glucosylceramide (GlcCer), which is partly converted by acid ceramidase to glucosylsphingosine (GlcSph) in the lysosome. Chronically elevated blood and tissue GlcSph is thought to contribute to symptoms in GD patients as well as to increased risk for Parkinson’s disease. On the other hand, formation of GlcSph may be beneficial since the water soluble sphingoid base is excreted via urine and bile. To study the role of excessive GlcSph formation during glucocerebrosidase deficiency, we studied zebrafish that have two orthologs of acid ceramidase, Asah1a and Asah1b. Only the latter is involved in the formation of GlcSph in glucocerebrosidase-deficient zebrafish as revealed by knockouts of Asah1a or Asah1b with glucocerebrosidase deficiency (either pharmacologically induced or genetic). Comparison of zebrafish with excessive GlcSph (gba1-/- fish) and without GlcSph (gba1-/-:asah1b-/- fish) allowed us to study the consequences of chronic high levels of GlcSph. Prevention of excessive GlcSph in gba1-/-:asah1b-/- fish did not restrict storage cells, GlcCer accumulation, or neuroinflammation. However, GD fish lacking excessive GlcSph show an ameliorated course of disease reflected by significantly increased lifespan, delayed locomotor abnormality, and delayed development of an abnormal curved back posture. The loss of tyrosine hydroxylase 1 (th1) mRNA, a marker of dopaminergic neurons, is slowed down in brain of GD fish lacking excessive GlcSph. In conclusion, in the zebrafish GD model, excess GlcSph has little impact on (neuro)inflammation or the presence of GlcCer-laden macrophages but rather seems harmful to th1-positive dopaminergic neurons.

Preclinical pharmacology of glucosylceramide synthase inhibitor venglustat in a GBA-related synucleinopathy model

Mutations in GBA, the gene encoding the lysosomal enzyme glucocerebrosidase (GCase), represent the greatest genetic risk factor for developing synucleinopathies including Parkinson’s disease (PD). Additionally, PD patients harboring a mutant GBA allele present with an earlier disease onset and an accelerated disease progression of both motor and non-motor symptoms. Preclinical studies in mouse models of synucleinopathy suggest that modulation of the sphingolipid metabolism pathway via inhibition of glucosylceramide synthase (GCS) using a CNS-penetrant small molecule may be a potential treatment for synucleinopathies. Here, we aim to alleviate the lipid storage burden by inhibiting the de novo synthesis of the primary glycosphingolipid substrate of GCase, glucosylceramide (GlcCer). We have previously shown that systemic GCS inhibition reduced GlcCer and glucosylsphingosine (GlcSph) accumulation, slowed α-synuclein buildup in the hippocampus, and improved cognitive deficits. Here, we studied the efficacy of a brain-penetrant clinical candidate GCS inhibitor, venglustat, in mouse models of GBA-related synucleinopathy, including a heterozygous Gba mouse model which more closely replicates the typical GBA-PD patient genotype. Collectively, these data support the rationale for modulation of GCase-related sphingolipid metabolism as a therapeutic strategy for treating GBA-related synucleinopathies.

Glucosylceramide and galactosylceramide, small glycosphingolipids with significant impact on health and disease.

Numerous clinical observations and exploitation of cellular and animal models indicate that glucosylceramide (GlcCer) and galactosylceramide (GalCer) are involved in many physiological and pathological phenomena. In many cases, the biological importance of these monohexosylcermides has been shown indirectly as the result of studies on enzymes involved in their synthesis and degradation. Under physiological conditions, GalCer plays a key role in the maintenance of proper structure and stability of myelin and differentiation of oligodendrocytes. On the other hand, GlcCer is necessary for the proper functions of epidermis. Such an important lysosomal storage disease as Gaucher disease (GD) and a neurodegenerative disorder as Parkinson’s disease are characterized by mutations in the GBA1 gene, decreased activity of lysosomal GBA1 glucosylceramidase and accumulation of GlcCer. In contrast, another lysosomal disease, Krabbe disease, is associated with mutations in the GALC gene, resulting in deficiency or decreased activity of lysosomal galactosylceramidase and accumulation of GalCer and galactosylsphingosine. Little is known about the role of both monohexosylceramides in tumor progression; however, numerous studies indicate that GlcCer and GalCer play important roles in the development of multidrug-resistance by cancer cells. It was shown that GlcCer is able to provoke immune reaction and acts as a self-antigen in GD. On the other hand, GalCer was recognized as an important cellular receptor for HIV-1. Altogether, these two molecules are excellent examples of how slight differences in chemical composition and molecular conformation contribute to profound differences in their physicochemical properties and biological functions.

Synthetic mRNA-based differentiation method enables early detection of Parkinson's phenotypes in neurons derived from Gaucher disease-induced pluripotent stem cells.

Gaucher disease, the most prevalent metabolic storage disorder, is caused by mutations in the glucocerebrosidase gene GBA1, which lead to the accumulation of glucosylceramide (GlcCer) in affected cells. Gaucher disease type 1 (GD1), although defined as a nonneuronopathic subtype, is accompanied by an increased risk of Parkinson's disease. To gain insights into the association of progressive accumulation of GlcCer and the Parkinson's disease phenotypes, we generated dopaminergic (DA) neurons from induced pluripotent stem cells (iPSCs) derived from a GD1 patient and a healthy donor control, and measured GlcCer accumulation by liquid chromatography‐mass spectrometry. We tested two DA neuron differentiation methods: a well‐established method that mimics a step‐wise developmental process from iPSCs to neural progenitor cells, and to DA neurons; and a synthetic mRNA‐based method that overexpresses a transcription factor in iPSCs. GD1‐specific accumulation of GlcCer was detected after 60 days of differentiation by the former method, whereas it was detected after only 10 days by the latter method. With this synthetic mRNA‐based rapid differentiation method, we found that the metabolic defect in GD1 patient cells can be rescued by the overexpression of wild‐type GBA1 or the treatment with an inhibitor for GlcCer synthesis. Furthermore, we detected the increased phosphorylation of α‐synuclein, a biomarker for Parkinson's disease, in DA neurons derived from a GD1 patient, which was significantly decreased by the overexpression of wild‐type GBA1. These results suggest that synthetic mRNA‐based method accelerates the analyses of the pathological mechanisms of Parkinson's disease in GD1 patients and possibly facilitates drug discovery processes.

The sirtuin inhibitor cambinol reduces intracellular glucosylceramide with ceramide accumulation by inhibiting glucosylceramide synthase.

The accumulation of glucosylceramide (GlcCer), which is synthesized by UDP-glucose ceramide glucosyltransferase (UGCG), is associated with several diseases, including Gaucher disease and Parkinson's disease. Since the inhibition of UGCG can be used to treat diseases caused by GlcCer accumulation, several UGCG inhibitors have been developed. In this study, we report on the inhibition of UGCG activity by cambinol, a sirtuin inhibitor. Unlike conventional UGCG inhibitors, cambinol has no structural similarity to GlcCer. LC-ESI MS/MS analysis revealed that the cellular GlcCer levels were reduced by cambinol with an increase in ceramide, the GlcCer precursor. Histidine 193 plays an important role in the inhibition of UGCG via a known UGCG inhibitor, D-PDMP. However, cambinol was found to inhibit UGCG activity in a histidine 193-independent manner. This study provides insights into the mechanism of inhibition of UGCG activity by cambinol, and provides a basis for the development of a cambinol-based novel UGCG inhibitor.

Glucocerebrosidase: Functions in and Beyond the Lysosome.

Glucocerebrosidase (GCase) is a retaining β-glucosidase with acid pH optimum metabolizing the glycosphingolipid glucosylceramide (GlcCer) to ceramide and glucose. Inherited deficiency of GCase causes the lysosomal storage disorder named Gaucher disease (GD). In GCase-deficient GD patients the accumulation of GlcCer in lysosomes of tissue macrophages is prominent. Based on the above, the key function of GCase as lysosomal hydrolase is well recognized, however it has become apparent that GCase fulfills in the human body at least one other key function beyond lysosomes. Crucially, GCase generates ceramides from GlcCer molecules in the outer part of the skin, a process essential for optimal skin barrier property and survival. This review covers the functions of GCase in and beyond lysosomes and also pays attention to the increasing insight in hitherto unexpected catalytic versatility of the enzyme.

Role of μ-glucosidase 2 in aberrant glycosphingolipid metabolism: model of glucocerebrosidase deficiency in zebrafish

β-glucosidases [GBA1 (glucocerebrosidase) and GBA2] are ubiquitous essential enzymes. Lysosomal GBA1 and cytosol-facing GBA2 degrade glucosylceramide (GlcCer); GBA1 deficiency causes Gaucher disease, a lysosomal storage disorder characterized by lysosomal accumulation of GlcCer, which is partly converted to glucosylsphingosine (GlcSph). GBA1 and GBA2 also may transfer glucose from GlcCer to cholesterol, yielding glucosylated cholesterol (GlcChol). Here, we aimed to clarify the role of zebrafish Gba2 in glycosphingolipid metabolism during Gba1 deficiency in zebrafish (Danio rerio), which are able to survive total Gba1 deficiency. We developed Gba1 (gba1-/-), Gba2 (gba2-/-), and double (gba1-/-:gba2-/-) zebrafish knockouts using CRISPR/Cas9 and explored the effects of both genetic and pharmacological interventions on GlcCer metabolism in individual larvae. Activity-based probes and quantification of relevant glycolipid metabolites confirmed enzyme deficiency. GlcSph increased in gba1-/- larvae (0.09 pmol/fish) but did not increase more in gba1-/-:gba2-/- larvae. GlcCer was comparable in gba1-/- and WT larvae but increased in gba2-/- and gba1-/-:gba2-/- larvae. Independent of Gba1 status, GlcChol was low in all gba2-/- larvae (0.05 vs. 0.18 pmol/fish in WT). Pharmacologic inactivation of zebrafish Gba1 comparably increased GlcSph. Inhibition of GlcCer synthase (GCS) in Gba1-deficient larvae reduced GlcCer and GlcSph, and concomitant inhibition of GCS and Gba2 with iminosugars also reduced excessive GlcChol. Finally, overexpression of human GBA1 and injection of recombinant GBA1 both decreased GlcSph. We determined that zebrafish larvae offer an attractive model to study glucosidase actions in glycosphingolipid metabolism in vivo, and we identified distinguishing characteristics of zebrafish Gba2 deficiency.

Brain-penetrant heat shock protein amplifier arimoclomol enhances GCase activity in in vitro Gaucher disease models

Brain-penetrant heat shock protein amplifier arimoclomol enhances GCase activity in in vitro Gaucher disease models

Biochemical Characterization of the GBA2 c.1780G>C Missense Mutation in Lymphoblastoid Cells from Patients with Spastic Ataxia.

The GBA2 gene encodes the non-lysosomal glucosylceramidase (NLGase), an enzyme that catalyzes the conversion of glucosylceramide (GlcCer) to ceramide and glucose. Mutations in GBA2 have been associated with the development of neurological disorders such as autosomal recessive cerebellar ataxia, hereditary spastic paraplegia, and Marinesco-Sjogren-Like Syndrome. Our group has previously identified the GBA2 c.1780G>C [p.Asp594His] missense mutation, in a Cypriot consanguineous family with spastic ataxia. In this study, we carried out a biochemical characterization of lymphoblastoid cell lines (LCLs) derived from three patients of this family. We found that the mutation strongly reduce NLGase activity both intracellularly and at the plasma membrane level. Additionally, we observed a two-fold increase of GlcCer content in LCLs derived from patients compared to controls, with the C16 lipid being the most abundant GlcCer species. Moreover, we showed that there is an apparent compensatory effect between NLGase and the lysosomal glucosylceramidase (GCase), since we found that the activity of GCase was three-fold higher in LCLs derived from patients compared to controls. We conclude that the c.1780G>C mutation results in NLGase loss of function with abolishment of the enzymatic activity and accumulation of GlcCer accompanied by a compensatory increase in GCase.

Fluorescence-Quenched Substrates for Quantitative Live Cell Imaging of Glucocerebrosidase Activity.

Glucocerebrosidase (GCase) is a lysosomal glycoside hydrolase that cleaves the glycolipid glucosylceramide (GlcCer). Deficiencies of this enzyme lead to accumulation of GlcCer and the development of the lysosomal storage disease known as Gaucher's disease. Recently, loss-of-function mutations in the GBA1 gene that encodes GCase have been linked to Parkinson's disease. Currently pursued therapeutic strategies to increase GCase involve enzyme replacement therapy, chemical chaperone therapy, and GCase activators. A challenge associated with advancing such strategies is to efficiently monitor GCase activity within the lysosomes of live cells. In this chapter, we review the design and use of the fluorescent-quenched probe GBA1-FQ2 to quantitatively measure GCase activity in lysosomes of live cells.

Glucosylceramide and Glucosylsphingosine Quantitation by Liquid Chromatography-Tandem Mass Spectrometry to Enable In Vivo Preclinical Studies of Neuronopathic Gaucher Disease.

Gaucher disease (GD) is caused by mutations in the GBA1 gene that encodes the lysosomal enzyme acid β-glucosidase (GCase). Reduced GCase activity primarily leads to the accumulation of two substrates, glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Current treatment options have not been shown to ameliorate the neurological pathology observed in the most severe forms of GD, clearly representing an unmet medical need. To better understand the relationship between GlcCer and GlcSph accumulation and ultimately their connection with the progression of neurological pathology, we developed LC-MS/MS methods to quantify GlcCer and GlcSph in mouse brain tissue. A significant challenge in developing these methods was the chromatographic separation of GlcCer and GlcSph from the far more abundant isobaric galactosyl epimers naturally occurring in white matter. After validation of both methods, we evaluated the levels of both substrates in five different GD mouse models, and found significant elevation of brain GlcSph in all five, while GlcCer was elevated in only one of the five models. In addition, we measured GlcCer and GlcSph levels in the brains of wild-type mice after administration of the GCase inhibitor conduritol β-epoxide (CBE), as well as the nonlysosomal β-glucosidase (GBA2) inhibitor N-butyldeoxygalactonojirimycin (NB-DGJ). Inhibition of GCase by CBE resulted in elevation of both sphingolipids; however, inhibition of GBA2 by NB-DGJ resulted in elevation of GlcCer only. Taken together, these data support the idea that GlcSph is a more selective and sensitive biomarker than GlcCer for neuronopathic GD in preclinical models.

Permeability Barrier and Microstructure of Skin Lipid Membrane Models of Impaired Glucosylceramide Processing

Ceramide (Cer) release from glucosylceramides (GlcCer) is critical for the formation of the skin permeability barrier. Changes in β-glucocerebrosidase (GlcCer’ase) activity lead to diminished Cer, GlcCer accumulation and structural defects in SC lipid lamellae; however, the molecular basis for this impairment is not clear. We investigated impaired GlcCer-to-Cer processing in human Cer membranes to determine the physicochemical properties responsible for the barrier defects. Minor impairment (5–25%) of the Cer generation from GlcCer decreased the permeability of the model membrane to four markers and altered the membrane microstructure (studied by X-ray powder diffraction and infrared spectroscopy), in agreement with the effects of topical GlcCer in human skin. At these concentrations, the accumulation of GlcCer was a stronger contributor to this disturbance than the lack of human Cer. However, replacement of 50–100% human Cer by GlcCer led to the formation of a new lamellar phase and the maintenance of a rather good barrier to the four studied permeability markers. These findings suggest that the major cause of the impaired water permeability barrier in complete GlcCer’ase deficiency is not the accumulation of free GlcCer but other factors, possibly the retention of GlcCer bound in the corneocyte lipid envelope.

Induced Pluripotent Stem Cell Modeling of Gaucher's Disease: What Have We Learned?

Gaucher’s disease (GD) is the most frequently inherited lysosomal storage disease, presenting both visceral and neurologic symptoms. Mutations in acid β-glucocerebrosidase disrupt the sphingolipid catabolic pathway promoting glucosylceramide (GlcCer) accumulation in lysosomes. Current treatment options are enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). However, neither of these approaches is effective in treating the neurological aspect of the disease. The use of small pharmacological compounds that act as molecular chaperones is a promising approach that is still experimental. In recent years, an association between GD and Parkinson like synucleinopathies has been discovered. Since 1992, a number of mouse models of GD have been the developed and partially reproduce phenotype of the disease. More recently, the discovery of direct reprograming has allowed the derivation of induced pluripotent stem cells (iPSc) from fibroblasts obtained from GD patients. iPSc can be expanded indefinitely in vitro and differentiated to macrophages and neurons, the main relevant cell types involved in GD. In this work, we review iPSc models of GD and summarize what we have learned from this system.

Identification of a feedback loop involving β-glucosidase 2 and its product sphingosine sheds light on the molecular mechanisms in Gaucher disease

The lysosomal acid β-glucosidase GBA1 and the non-lysosomal β-glucosidase GBA2 degrade glucosylceramide (GlcCer) to glucose and ceramide in different cellular compartments. Loss of GBA2 activity and the resulting accumulation of GlcCer results in male infertility, whereas mutations in the GBA1 gene and loss of GBA1 activity cause the lipid-storage disorder Gaucher disease. However, the role of GBA2 in Gaucher disease pathology and its relationship to GBA1 is not well understood. Here, we report a GBA1-dependent down-regulation of GBA2 activity in patients with Gaucher disease. Using an experimental approach combining cell biology, biochemistry, and mass spectrometry, we show that sphingosine, the cytotoxic metabolite accumulating in Gaucher cells through the action of GBA2, directly binds to GBA2 and inhibits its activity. We propose a negative feedback loop, in which sphingosine inhibits GBA2 activity in Gaucher cells, preventing further sphingosine accumulation and, thereby, cytotoxicity. Our findings add a new chapter to the understanding of the complex molecular mechanism underlying Gaucher disease and the regulation of β-glucosidase activity in general.

Mactosylceramide prevents glial cell overgrowth by inhibiting insulin and fibroblast growth factor receptor signaling.

Receptor tyrosine kinase (RTK) signaling controls key aspects of cellular differentiation, proliferation, survival, metabolism, and migration. Deregulated RTK signaling also underlies many cancers. Glycosphingolipids (GSL) are essential elements of the plasma membrane. By affecting clustering and activity of membrane receptors, GSL modulate signal transduction, including that mediated by the RTK. GSL are abundant in the nervous system, and glial development in Drosophila is emerging as a useful model for studying how GSL modulate RTK signaling. Drosophila has a simple GSL biosynthetic pathway, in which the mannosyltransferase Egghead controls conversion of glucosylceramide (GlcCer) to mactosylceramide (MacCer). Lack of elongated GSL in egghead (egh) mutants causes overgrowth of subperineurial glia (SPG), largely due to aberrant activation of phosphatidylinositol 3-kinase (PI3K). However, to what extent this effect involves changes in upstream signaling events is unresolved. We show here that glial overgrowth in egh is strongly linked to increased activation of Insulin and fibroblast growth factor receptors (FGFR). Glial hypertrophy is phenocopied when overexpressing gain-of-function mutants of the Drosophila insulin receptor (InR) and the FGFR homolog Heartless (Htl) in wild type SPG, and is suppressed by inhibiting Htl and InR activity in egh. Knockdown of GlcCer synthase in the SPG fails to suppress glial overgrowth in egh nerves, and slightly promotes overgrowth in wild type, suggesting that RTK hyperactivation is caused by absence of MacCer and not by GlcCer accumulation. We conclude that an early product in GSL biosynthesis, MacCer, prevents inappropriate activation of insulin and fibroblast growth factor receptors in Drosophila glia.

AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides

The membrane glycolipid glucosylceramide (GlcCer) plays a critical role in cellular homeostasis. Its intracellular levels are thought to be tightly regulated. How cells regulate GlcCer levels remains to be clarified. AMP-activated protein kinase (AMPK), which is a crucial cellular energy sensor, regulates glucose and lipid metabolism to maintain energy homeostasis. Here, we investigated whether AMPK affects GlcCer metabolism. AMPK activators (5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and metformin) decreased intracellular GlcCer levels and synthase activity in mouse fibroblasts. AMPK inhibitors or AMPK siRNA reversed these effects, suggesting that GlcCer synthesis is negatively regulated by an AMPK-dependent mechanism. Although AMPK did not affect the phosphorylation or expression of GlcCer synthase, the amount of UDP-glucose, an activated form of glucose required for GlcCer synthesis, decreased under AMPK-activating conditions. Importantly, the UDP-glucose pyrophosphatase Nudt14, which degrades UDP-glucose, generating UMP and glucose 1-phosphate, was phosphorylated and activated by AMPK. On the other hand, suppression of Nudt14 by siRNA had little effect on UDP-glucose levels, indicating that mammalian cells have an alternative UDP-glucose pyrophosphatase that mainly contributes to the reduction of UDP-glucose under AMPK-activating conditions. Because AMPK activators are capable of reducing GlcCer levels in cells from Gaucher disease patients, our findings suggest that reducing GlcCer through AMPK activation may lead to a new strategy for treating diseases caused by abnormal accumulation of GlcCer.