Assay of ß-glucosidase 2 (GBA2) activity using lithocholic acid ß-3-O-glucoside substrate for cultured fibroblasts and glucosylceramide for brain tissue.

Beta (ß)-glucosidase 2 (GBA2) is deficient in a form of human spastic paraplegia due to defects in GBA2 (SPG46). GBA2 was proposed as a modifier of Gaucher disease, a lysosomal storage disease resulting from deficient ß-glucosidase 1; GBA1. Current GBA2 activity assays using artificial substrates incompletely model the activity encountered in vivo. We studied GBA2 activity, using lithocholic acid ß-glucoside or glucosylceramide as natural ß-glucosidase substrates in murine tissues or cultured patient fibroblasts with the pathologic genotypes: Gba1-/-; Gba2-/-; GBA1-/-; GBA2+/- and found expected and unexpected deviations from normal controls.

Reducing GBA2 Activity Ameliorates Neuropathology in Niemann-Pick Type C Mice.

The enzyme glucocerebrosidase (GBA) hydrolyses glucosylceramide (GlcCer) in lysosomes. Markedly reduced GBA activity is associated with severe manifestations of Gaucher disease including neurological involvement. Mutations in the GBA gene have recently also been identified as major genetic risk factor for Parkinsonism. Disturbed metabolism of GlcCer may therefore play a role in neuropathology. Besides lysosomal GBA, cells also contain a non-lysosomal glucosylceramidase (GBA2). Given that the two ß-glucosidases share substrates, we speculated that over-activity of GBA2 during severe GBA impairment might influence neuropathology. This hypothesis was studied in Niemann-Pick type C (Npc1-/-) mice showing secondary deficiency in GBA in various tissues. Here we report that GBA2 activity is indeed increased in the brain of Npc1-/- mice. We found that GBA2 is particularly abundant in Purkinje cells (PCs), one of the most affected neuronal populations in NPC disease. Inhibiting GBA2 in Npc1-/- mice with a brain-permeable low nanomolar inhibitor significantly improved motor coordination and extended lifespan in the absence of correction in cholesterol and ganglioside abnormalities. This trend was recapitulated, although not to full extent, by introducing a genetic loss of GBA2 in Npc1-/- mice. Our findings point to GBA2 activity as therapeutic target in NPC.

High ß-glucosidase (GBA) activity not attributable to GBA1 and GBA2 in live normal and enzyme-deficient fibroblasts may emphasise the role of additional GBAs.

Beta-glucosidases (GBA) include GBA1, GBA2 and other ß-glucosidases (non-GBA1-2). GBA1 is a lysosomal and GBA2 an extra-lysosomal enzyme. GBA1- and GBA2-deficient genetic conditions, with different phenotypes, are glucosylceramide (GC; the main GBA substrate) accumulating diseases. To study the activity profile of GBA, live fibroblasts were loaded with radioactive GC. The GC metabolism was measured in wild-type, GBA1-deficient (Gaucher disease) and GBA2-deficient (Gba2(-/- )mouse) cells. The differences found allowed the prediction of marked proportions of GBA1, GBA2, and particularly non-GBA1-2 (probably including GBA3, a cytosolic ß-glucosidase) activity for wild-type cells. The high proportion of non-GBA1-2 suggests an important role of these enzymes.

Accumulation of glucosylceramide in the absence of the beta-glucosidase GBA2 alters cytoskeletal dynamics.

Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types.

Potential risk factors for patient mortality during admission to the intensive care units.

OBJECTIVE: To determine the prognostic importance of admission time, admission day, admission source, and route of admission, attached devices before admission, diagnosis of patients, and total Simplified Acute Physiology Score (SAPS) II score during admission on intensive care unit mortality rates. METHODS: This retrospective study was carried out with 545 patients admitted to adult intensive care units between January 2006 and January 2011 at Zonguldak, Turkey. Computerised database and patient records were used for data collection. Data from the patient records was collected by a constructed survey form and SAPS II scale. The data was evaluated by numeric values and percentages, Pearson correlation analysis, logistic regression analysis, Chi-square, and student t-tests. RESULTS: The mortality rate was 60.4%. The patients who died had a higher SAPS II score. The mortality rate was higher in admissions from the emergency department (p=0.000), admissions of patients on a stretcher (p=0.000), the existence of an intubation tube (p=0.000) and monitor (p=0.001), and in patients with respiratory tract illness (p=0.000), and infection (p=0.000). A significant difference was not found between the admission day (p=0.761), and time (p=0.063). CONCLUSION: The SAPS II scores of the patients, the route of admission and admission source, being intubated and connected to a monitor, admission diagnosis, and length of stay in intensive care units was increased to mortality rate. However, the days and hours of admission did not significantly affect mortality.

1-O-acylceramides are natural components of human and mouse epidermis.

The lipid-rich stratum corneum functions as a barrier against pathogens and desiccation inter alia by an unbroken meshwork of extracellular lipid lamellae. These lamellae are composed of cholesterol, fatty acids, and ceramides (Cers) in an equimolar ratio. The huge class of skin Cers consists of three groups: group I, "classical" long and very long chain Cers; group II, ultra-long chain Cers; and group III, ω-esterified ultra-long chain Cers, which are esterified either with linoleic acid or with cornified envelope proteins and are required for the water permeability barrier. Here, we describe 1-O-acylceramides as a new class of epidermal Cers in humans and mice. These Cers contain, in both the N- and 1-O-position, long to very long acyl chains. They derive from the group I of classical Cers and make up 5% of all esterified Cers. Considering their chemical structure and hydrophobicity, we presume 1-O-acylceramides to contribute to the water barrier homeostasis. Biosynthesis of 1-O-acylceramides is not dependent on lysosomal phospholipase A2. However, glucosylceramide synthase deficiency was followed by a 7-fold increase of 1-O-acylceramides, which then contributed 30% to all esterified Cers. Furthermore, loss of neutral glucosylceramidase resulted in decreased levels of a 1-O-acylceramide subgroup. Therefore, we propose 1-O-acylceramides to be synthesized at endoplasmic reticulum-related sites.

Functional and genetic characterization of the non-lysosomal glucosylceramidase 2 as a modifier for Gaucher disease.

BACKGROUND: Gaucher disease (GD) is the most common inherited lysosomal storage disorder in humans, caused by mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GBA1). GD is clinically heterogeneous and although the type of GBA1 mutation plays a role in determining the type of GD, it does not explain the clinical variability seen among patients. Cumulative evidence from recent studies suggests that GBA2 could play a role in the pathogenesis of GD and potentially interacts with GBA1. METHODS: We used a framework of functional and genetic approaches in order to further characterize a potential role of GBA2 in GD. Glucosylceramide (GlcCer) levels in spleen, liver and brain of GBA2-deficient mice and mRNA and protein expression of GBA2 in GBA1-deficient murine fibroblasts were analyzed. Furthermore we crossed GBA2-deficient mice with conditional Gba1 knockout mice in order to quantify the interaction between GBA1 and GBA2. Finally, a genetic approach was used to test whether genetic variation in GBA2 is associated with GD and/ or acts as a modifier in Gaucher patients. We tested 22 SNPs in the GBA2 and GBA1 genes in 98 type 1 and 60 type 2/3 Gaucher patients for single- and multi-marker association with GD. RESULTS: We found a significant accumulation of GlcCer compared to wild-type controls in all three organs studied. In addition, a significant increase of Gba2-protein and Gba2-mRNA levels in GBA1-deficient murine fibroblasts was observed. GlcCer levels in the spleen from Gba1/Gba2 knockout mice were much higher than the sum of the single knockouts, indicating a cross-talk between the two glucosylceramidases and suggesting a partially compensation of the loss of one enzyme by the other. In the genetic approach, no significant association with severity of GD was found for SNPs at the GBA2 locus. However, in the multi-marker analyses a significant result was detected for p.L444P (GBA1) and rs4878628 (GBA2), using a model that does not take marginal effects into account. CONCLUSIONS: All together our observations make GBA2 a likely candidate to be involved in GD etiology. Furthermore, they point to GBA2 as a plausible modifier for GBA1 in patients with GD.

The non-lysosomal ß-glucosidase GBA2 is a non-integral membrane-associated protein at the endoplasmic reticulum (ER) and Golgi.

GBA1 and GBA2 are both ß-glucosidases, which cleave glucosylceramide (GlcCer) to glucose and ceramide. GlcCer is a main precursor for higher order glycosphingolipids but might also serve as intracellular messenger. Mutations in the lysosomal GBA1 underlie Gaucher disease, the most common lysosomal storage disease in humans. Knocking out the non-lysosomal GBA2 in mice results in accumulation of GlcCer outside the lysosomes in various tissues (e.g. testis and liver) and impairs sperm development and liver regeneration. However, the underlying mechanisms are not well understood. To reveal the physiological function of GBA2 and, thereby, of the non-lysosomal GlcCer pool, it is important to characterize the localization of GBA2 and its activity in different tissues. Thus, we generated GBA2-specific antibodies and developed an assay that discriminates between GBA1 and GBA2 without the use of detergent. We show that GBA2 is not, as previously thought, an integral membrane protein but rather a cytosolic protein that tightly associates with cellular membranes. The interaction with the membrane, in particular with phospholipids, is important for its activity. GBA2 is localized at the ER and Golgi, which puts GBA2 in a key position for a lysosome-independent route of GlcCer-dependent signaling. Furthermore, our results suggest that GBA2 might affect the phenotype of Gaucher disease, because GBA2 activity is reduced in Gba1 knock-out fibroblasts and fibroblasts from a Gaucher patient. Our results provide the basis to understand the mechanism for GBA2 function in vivo and might help to unravel the role of GBA2 during pathogenesis of Gaucher disease.

Inhibition of hepatitis C virus RNA translation by antisense bile acid conjugated phosphorothioate modified oligodeoxynucleotides (ODN).

BACKGROUND: The 5'-noncoding region (5'NCR) of the HCV-genome comprises an internal ribosome entry site essential for HCV-translation/replication. Phosphorothioate oligodeoxynucleotides (tS-ODN) complementary to this region can inhibit HCV-translation in vitro. In this study, bile acid conjugated tS-ODN were generated to increase cell-selective inhibition of 5'NCR-dependent HCV-translation. METHODS: Different bile acid conjugated tS-ODN complementary to the HCV5'NCR were selected for their inhibitory potential in an in vitro transcription/translation assay. To analyze OATP (organic anion transporting polypeptides)-selective uptake of bile acid conjugated ODN, different hepatoma cells were stably transfected with the OATP1B1-transporter and primary human hepatocytes were used. An adenovirus encoding the HCV5'NCR fused to the luciferase gene (Ad-GFP-NCRluc) was generated to quantify 5'NCR-dependent HCV gene expression in OATP-overexpressing hepatoma cells and in vivo. RESULTS: A 17mer phosphorothioate modified ODN (tS-ODN4_13) complementary to HCV5'NCR was able to inhibit 5'NCR-dependent HCV-translation in an in vitro transcription/translation test system by more than 90% and it was also effective in Huh7-cells containing the HCV subgenomic replicon. Conjugation to taurocholate (tS-ODN4_13T) significantly increased selective ODN uptake by primary human hepatocytes and by OATP1B1-expressing HepG2-cells compared to parental HepG2-cells. Correspondingly, tS-ODN4_13T significantly inhibited HCV gene expression in liver-derived OATP1B1-expressing HepG2- or CCL13-cells up to 70% compared to unconjugated tS-ODN and compared to mismatch taurocholate coupled tS-ODN. In vivo, tS-ODN4_13T showed also a trend to block 5'NCR-dependent HCV gene expression. CONCLUSIONS: The tested taurocholate conjugated 17mer antisense ODN complementary to HCV5'NCV showed an increased and selective uptake by hepatocytes and liver-derived cells through OATP-mediated transport resulting in enhanced specific inhibition of HCV gene expression in vitro and in vivo. Thus, this novel approach may represent a promising strategy to improve antisense approaches with ODN in the control of hepatitis C infection.

Beta-glucosidase 2 knockout mice with increased glucosylceramide show impaired liver regeneration.

BACKGROUND AND AIMS: Glycolipids have been shown to serve specialized functions in cell signalling, proliferation and differentiation processes, which are all important during liver regeneration. We previously generated beta-glucosidase 2 (GBA2) knockout mice that accumulate the glycolipid glucosylceramide in various tissues, including the liver. The present study addressed the role of GBA2-deficiency and subsequent glucosylceramide accumulation in liver regeneration. METHODS: Gba2 knockout and wild-type mice were subjected to two-third partial hepatectomy. Mice were sacrificed at different time points, blood was collected, and the remnant liver was removed. Glucosylceramide and ceramide were quantified using mass spectrometry from whole liver and isolated hepatocytes. Serum and hepatocytic supernatant of IL-6, TNF-α and TGF-ß levels were measured using ELISA. Cell signalling proteins were analysed using immunoblots. RESULTS: Regenerating liver after partial hepatectomy showed a significant increase of hepatic glucosylceramide in GBA2-deficient mice compared to controls. Accumulation of glucosylceramide was associated with a delay in liver regeneration and reduced serum levels of IL-6 and TNF-α. Furthermore, reduced IL-6 led to decreased expression of the phosphorylated form of the signal transducer and activator of transcription 3 (P-STAT3). CONCLUSIONS: We conclude that increased glucosylceramide affects cytokine- and growth factor-mediated signalling pathways during liver regeneration. Thus, the repression of IL-6/STAT3 signalling pathway seems to be one of the mechanisms for the delay of liver regeneration in GBA2-deficient mice.

Beta-glucosidase 1 (GBA1) is a second bile acid ß-glucosidase in addition to ß-glucosidase 2 (GBA2). Study in ß-glucosidase deficient mice and humans.

Beta-glucosidase 1 (GBA1; lysosomal glucocerebrosidase) and ß-glucosidase 2 (GBA2, non-lysosomal glucocerebrosidase) both have glucosylceramide as a main natural substrate. The enzyme-deficient conditions with glucosylceramide accumulation are Gaucher disease (GBA-/- in humans), modelled by the Gba-/- mouse, and the syndrome with male infertility in the Gba2-/- mouse, respectively. Before the leading role of glucosylceramide was recognised for both deficient conditions, bile acid-3-O-ß-glucoside (BG), another natural substrate, was viewed as the main substrate of GBA2. Given that GBA2 hydrolyses both BG and glucosylceramide, it was asked whether vice versa GBA1 hydrolyses both glucosylceramide and BG. Here we show that GBA1 also hydrolyses BG. We compared the residual BG hydrolysing activities in the GBA1-/-, Gba1-/- conditions (where GBA2 is the almost only active ß-glucosidase) and those in the Gba2-/- condition (GBA1 active), with wild-type activities, but we used also the GBA1 inhibitor isofagomine. GBA1 and GBA2 activities had characteristic differences between the studied fibroblast, liver and brain samples. Independently, the hydrolysis of BG by pure recombinant GBA1 was shown. The fact that both GBA1 and GBA2 are glucocerebrosidases as well as bile acid ß-glucosidases raises the question, why lysosomal accumulation of glucosylceramide in GBA1 deficiency, and extra-lysosomal accumulation in GBA2 deficiency, are not associated with an accumulation of BG in either condition.

Knock-out of ß-glucosidase 2 has no influence on dextran sulfate sodium-induced colitis.

BACKGROUND/AIMS: The non-lysosomal glucosylceramidase, ß-glucosidase (Gba2), hydrolyzes glucosylceramide to glucose and ceramide (Cer). Cer is a potent second-messenger lipid that plays an important role in signaling cascades involved in apoptosis. The aim of this study was to investigate whether Gba2 knock-out (Gba2(-/-)) affects the extent of dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: Acute colitis was induced in wild-type (WT) and Gba2(-/-) mice by administration of 2% DSS in drinking water. After 7 days, mice underwent colonoscopy and were sacrificed. RESULTS: Both DSS-treated WT (n = 10) and Gba2(-/-) (n = 12) mice showed elevated histological and endoscopic scores compared to respective H(2)O controls (n = 9 each). However, no significant differences between the DSS groups were detected. Flow cytometric analysis of propidium iodide staining, cleavage of caspases-3 and -8, indicative for apoptosis, as well as Cer levels were not altered in DSS-treated WT or Gba2(-/-) mice. Gba2(-/-) resulted in slightly decreased expression of glucocerebrosidase (Gba1) as well as in upregulation of proteins being involved in cellular regeneration, such as STAT3 (signal transducer and activator of transcription), JNK and iNOS, upon DSS treatment. CONCLUSION: We demonstrate that Gba2(-/-) does not affect the extent of DSS-induced inflammation in mice, however, it might be involved in tissue regeneration in response to toxic agents.

Inhibition of hepatitis C virus gene expression by adenoviral vectors encoding antisense RNA in vitro and in vivo.

BACKGROUND & AIMS: In this study, adenoviral vectors encoding an antisense RNA complementary to the 5' non-coding region (5'NCR) of the HCV-genome were generated to inhibit HCV-RNA gene expression in cell culture and in vivo. METHODS: First and second-generation (with E4-deletion) adenoviruses encoding the HCV5'NCR in antisense direction (Ad-NCRas and Ad-E4del-NCRas) were generated. Inhibition of HCV gene expression was analyzed in hepatoma cells stably transfected with the HCV5'NCR cDNA fused to the firefly luciferase gene (NCRluc), as well as in the HCV subgenomic replicon (genotypes 1b and 2a) and the fully infectious HCV JFH-1 cell culture systems. For in vivo experiments, an adenovirus encoding the NCRluc-gene was injected intravenously to achieve a NCR-dependent luciferase-expression in the liver of C3H/HeNcrl-mice. RESULTS: Forty eight hours after transduction with GFP-encoding adenoviruses, >85% of HepG2-, CCL13-and Huh7-cells expressed GFP. Surprisingly, GFP-expression of E4-deleted adenoviruses was considerably reduced at the same MOI. Using antisense first-generation adenoviruses (Ad-NCRas), a significant inhibition of the 5'NCR-dependent HCV-gene expression (54±19% in HepG2-cells and 66.2±15% in Huh7-cells) was achieved 48h after transduction. In Huh7-cells containing the HCV subgenomic replicons and in infectious HCV JFH-1 cell cultures, adenovirus-mediated transcription of antisense 5'NCR significantly blocked HCV-replication (40% and 76%, respectively). Corresponding to low transgene expression, the maximal inhibition reached with Ad-delE4-NCRas was 30%. In vivo, antisense adenoviral vectors also showed a significant inhibition (40%) of NCR-dependent luciferase expression compared to control adenoviruses (p<0.05). CONCLUSIONS: The data indicate that HCV gene expression can be inhibited by antisense RNA encoding adenoviruses in the tested settings.

CD40ligand-expressing dendritic cells induce regression of hepatocellular carcinoma by activating innate and acquired immunity in vivo.

UNLABELLED: Dendritic cells (DCs) are professional antigen-presenting cells able to prime T-cells against tumor-associated antigens (TAA), but their potential to induce hepatocellular carcinoma (HCC) regression is still limited. CD40/CD40L interaction is essential for DC activation and induction of antigen-specific T-cells. In this study, transduction of TAA-pulsed DC with a CD40L-encoding adenovirus (Ad-CD40L) was used to improve the immune response induced by DC toward HCC. Bone marrow-derived DC from C3H/HeNcrl mice were cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4. On day 6, tumor-lysate pulsed DCs were infected with adenoviruses. HCCs were induced by inoculation of mice with Hepa129-cells subcutaneously. When tumor-volume was 100 to 400 mm(3), DCs were injected intratumorally, subcutaneously, or intravenously. Ad-CD40L transduction exerted CD40/CD40L interactions between DCs, increasing DC immunostimulation with up-regulation of CD80/CD86- and interleukin-12 (IL-12) expression. Intratumoral injection of CD40L-DC was superior to intravenous or subcutaneous treatments, yielding tumor elimination in almost 70% of mice. Moreover, all tumor-free animals were protected against hepatic tumor cell rechallenge. In a preventive setting, subcutaneous injection of CD40L-expressing DCs protected 50% of mice for more than 3 months toward tumor cell challenge. The induced immune response seemed to be dependent on cross-priming with Th1-lymphocytes in the lymph nodes, because transduced DCs were redetected in lymphoid tissues. In addition, immunohistochemistry of tumors indicated a significant tumor infiltration with CD4+, CD8+ T cells and natural killer (NK) cells. Tumor-infiltrating lymphocytes were tumor-specific, as shown in interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot and T-cell proliferation assays. CONCLUSION: Transduction of DCs with Ad-CD40L increases significantly the stimulatory capacity of DCs. Intratumoral injection of DCs activates both acquired and innate immunity, inducing complete regression of established tumors and long-term immunity against tumor recurrence. This approach improves the antitumoral potential of DCs.

Accumulation of glucosylceramide in murine testis, caused by inhibition of beta-glucosidase 2: implications for spermatogenesis.

One of the hallmarks of male germ cell development is the formation of a specialized secretory organelle, the acrosome. This process can be pharmacologically disturbed in C57BL/6 mice, and thus infertility can be induced, by small molecular sugar-like compounds (alkylated imino sugars). Here the biochemical basis of this effect has been investigated. Our findings suggest that in vivo alkylated imino sugars primarily interact with the non-lysosomal glucosylceramidase. This enzyme cleaves glucosylceramide into glucose and ceramide, is sensitive to imino sugars in vitro, and has been characterized as beta-glucosidase 2 (GBA2). Imino sugars raised the level of glucosylceramide in brain, spleen, and testis, in a dose-dependent fashion. In testis, multiple species of glucosylceramide were similarly elevated, those having long acyl chains (C16-24), as well as those with very long polyunsaturated acyl chains (C28-30:5). Both of these GlcCer species were also increased in the testes from GBA2-deficient mice. When considering that the very long polyunsaturated sphingolipids are restricted to germ cells, these results indicate that in the testis GBA2 is present in both somatic and germ cells. Furthermore, in all mouse strains tested imino sugar treatment caused a rise in testicular glucosylceramide, even in a number of strains, of which the males remain fertile after drug administration. Therefore, it appears that acrosome formation can be derailed by accumulation of glucosylceramide in an extralysosomal localization, and that the sensitivity of male germ cells to glucosylceramide is genetically determined.

Mutation of beta-glucosidase 2 causes glycolipid storage disease and impaired male fertility.

beta-Glucosidase 2 (GBA2) is a resident enzyme of the endoplasmic reticulum thought to play a role in the metabolism of bile acid-glucose conjugates. To gain insight into the biological function of this enzyme and its substrates, we generated mice deficient in GBA2 and found that these animals had normal bile acid metabolism. Knockout males exhibited impaired fertility. Microscopic examination of sperm revealed large round heads (globozoospermia), abnormal acrosomes, and defective mobility. Glycolipids, identified as glucosylceramides by mass spectrometry, accumulated in the testes, brains, and livers of the knockout mice but did not cause obvious neurological symptoms, organomegaly, or a reduction in lifespan. Recombinant GBA2 hydrolyzed glucosylceramide to glucose and ceramide; the same reaction catalyzed by the beta-glucosidase acid 1 (GBA1) defective in subjects with the Gaucher's form of lysosomal storage disease. We conclude that GBA2 is a glucosylceramidase whose loss causes accumulation of glycolipids and an endoplasmic reticulum storage disease.