Accumulation Of Glucosylceramide Research Articles

Cerebrospinal Fluid Profiles in Parkinson's Disease: No Accumulation of Glucosylceramide, but Significant Downregulation of Active Complement C5 Fragment.

As mutations in glucocerebrosidase 1 (GBA1) are a major risk factor for Parkinson's disease (PD), decreased GBA1 activity might play an important role in the pathogenesis of the disease. However, there are currently no reports on glucosylceramide levels in the cerebrospinal fluid (CSF) in PD. We investigated whether glucosylceramide accumulation and abnormal immune status in the brain are associated with PD. We measured glucosylceramide by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) as well as levels of the active fragment of complement C5, C5a, in the CSF of 33 PD, 15 amyotrophic lateral sclerosis (ALS) and 22 neurologically normal control (NNC) subjects. Serum C5a levels in all PD and ALS cases and in a limited number of NNC subjects (n = 8) were also measured. C5a levels in CSF were significantly downregulated in PD compared with NNC. Moreover, CSF C5a/serum C5a ratio showed pronounced perturbations in PD and ALS patients. LC-ESI-MS/MS revealed a statistically significant accumulation of a specific subspecies of glucosylceramide (d18 : 1/C23 : 0 acyl chain fatty acid) in ALS, but not in PD. Interestingly, CSF glucosylceramide (d18 : 1/C23 : 0) exhibited a significant correlation with CSF C5a levels in PD, but not ALS. No correlation was observed between C5a levels or glucosylceramide subspecies content and disease duration, levodopa equivalent daily dose or Hoehn & Yahr staging in PD. Our findings demonstrate complement dysregulation without glucosylceramide accumulation in PD CSF. Furthermore, we found an association between a specific glucosylceramide subspecies and immune status in PD.

Polar Lipid Fraction from Golden Oyster Mushrooms (Pleurotus citrinopileatus) Suppresses Colon Injuries from Inflammatory Stresses in vivo and in vitro.

The rising incidence of inflammatory bowel disease (IBD) in East Asian countries has necessitated the implementation of preventive methods in the form of dietary supplementation and changes in dietary habits. We have previously reported that dietary golden oyster mushroom (Pleurotus citrinopileatus) ethanol extract (GOMEE) suppresses intestinal inflammation in mouse models of IBD induced by dextran sulfate sodium salt (DSS). Here, we investigated the components of GOMEE that exert suppressive effects on colon inflammation in vivo and in vitro. The total lipid fraction was extracted from GOMEE, and the polar and neutral lipid fractions were subsequently separated via solvent fractionation. Mice were assigned to dietary groups-control, 1% total lipid, 1% polar lipid, or 1% neutral lipid diet-and fed the respective diets for one week; mice were administered 1.5% DSS in drinking water ad libitum for 20 days. Dietary supplementation with the total or polar lipid fraction alleviated DSS-induced chorionic crypt injury as determined by morphological observation, while dietary supplementation with the neutral lipid fraction did not produce such effects. In the in vitro study, using differentiated Caco-2 cells as the colon model, treatment with the total or polar lipid fraction suppressed cell decrease by lipopolysaccharide (LPS)-induced apoptosis whereas treatment with the neutral lipid fraction did not. Moreover, accumulation of glucosylceramide (GlcCer), a fungal sphingolipid, was observed in the intestinal cells after treatment with polar lipid fraction. These results suggest that the active components of GOMEE that suppress colon inflammation are polar lipids, especially GlcCer. The structure of mushroom GlcCer differs from that of the plant counterpart and is therefore expected to exert different food functions.

Decreased expression of GBA3 correlates with a poor prognosis in hepatocellular carcinoma patients.

Beta-glucosidase (GBA), also known as acid β-glucosidase, exhibits an activity of glucosylceramidase (EC 3.2.1.45). Three main isoforms of β-glucosidases have been identified in mammals: GBA1, GBA2, and GBA3. The deficiency of these enzymes leads to glucosylceramide accumulation, resulting in Gaucher's disease. The present study is focused on the cytosolic β-glucosidase, GBA3, and its relationship with hepatocellular carcinoma (HCC). The expression of GBA3 mRNA in HCC was evaluated first using the TCGA database, and then by immunohistochemistry using tissue microarrays of 328 clinically characterized HCC samples and 151 non-tumor liver controls. Moreover, the presence of a correlation between GBA3 expression and clinicopathological characteristics of patients was examined. The obtained results indicated that the expression of GBA3 mRNA was significantly lower in HCC than in the adjacent non-tumor liver tissues. The expression of GBA3 was inversely related to the number of tumors (p=0.041), tumor size (p<0.001), Edmondson grade (p=0.007), microvascular invasion (p=0.049), patient status (p<0.001), and α-fetoprotein level (p<0.001). Patients exhibiting low GBA3 expression had a shorter survival time than those with high expression (p<0.001). In conclusion, the decreased GBA3 expression is strongly associated with a poor prognosis in HCC patients, and GBA3 may be a potential therapeutic target for HCC.

Evaluation of in Vitro Macrophage Characterization in Gaucher Type 1 (GD1) Patients

Gaucher disease type 1 is the most common inherited lysosomal storage disorder caused by the deficiency of lysosomal β-glucocerebrosidase (GBA, acid-β-glucosidase), required for the degradation of glycosphingolipids. The deficiency of the enzyme results in the widespread accumulation of glucosylceramide in macrophages, leading to anemia, thrombocytopenia and coagulopathy, visceral (hepatosplenomegaly, lungs) and skeletal manifestations (deformities, fractures, avascular osteonecrosis). However, GD manifestations are caused not only by the burden of glucosylceramide storage, but also by macrophage activation. It seems that GD reflects the downstream consequences of inappropriate macrophage activation, with the release of pro-inflammatory cytokines and other responses to storage material. The aim of this study is to assess in vitro phenotypic characterization, functional properties and gene expression anaysis of GD1 macrophages in order to understand their possible role in inflammation and in impairment of iron metabolism occurring in GD patients. Monocytes were isolated from buffy coats of GD patients (n=3) and controls (n=3) by applying an in vitro model protocol. Monocytes were expanded in ImmunoCult™ medium and 1% ZellShield® antibiotic cocktail. Differentiation was induced in ImmunoCult™ medium, 50 ng/mL M-CSF and 50 ng/mL GM-CSF. To mimic the in vivo condition, macrophage population was loaded with erythrocytes ghosts isolated from GD patients. Cell morphology was analyzed on cytocentrifuged preparations stained with May-Grünwald Giemsa (MGG). Surface marker expression (CD11, CD33, CD68, CD64) were examined by flow cytometry to evaluate macrophages differentiation and phenotype. Gene expression analysis of iron metabolism-related genes was evaluated through Real-Time Quantitative PCR. Biochemical indices (NTBI, GDF15, sTfR and chitotriosidase) were analyzed in supernatant through ELISA assay. Flow cytometry analysis (Fig.1) revealed that without erythrocytes ghosts (preM∅), the proportion of CD11+/CD68+ macrophage population was similar between GD patients and control. However, in GD macrophages, when loaded with Gaucher erythrocytes ghosts (M∅+ghosts), the proportion of CD11++/CD68++ cells increased (36.4%), as a reflection of a more pro-inflammatory phenotype. Treated controls showed no differences. To further characterize these different macrophages subpopulations in GD patients, we used an additional parameter, CD64, because CD64/CD68 markers are specific for M1/M2 polarization. GD M∅+ghosts showed an higher percentage of CD64++/CD68++ population (78%) in comparison of GD pre-M∅ (45%) and controls (36%), confirming a M1 proinflammatory phenotype of GD macrophages loaded with erythrocytes ghosts. Under microscope evaluation, GD M∅+ghosts presented high number of spindle-shaped fibroblastoid, typically M1 phenotype cells, rather than large flat-round cells (M2 cells). Moreover, morphological staining of these cells confirmed the typical features of GD cells with basophilic cytoplasm with characteristic crinkles. Controls showed no differences in macrophage features when loaded with erythrocytes ghosts. To confirm the pro-inflammatory potential of GD M∅+ghosts, high levels of pro-inflammatory mediators TNF-α, IL-1β and (s)TfR) were found in supernatant of GD M∅+ghosts (72,9±8,5, 24,7±2,7 and 3,1±4,4, respectively) compared to pre-M∅ and controls. High GDF15 expression in GD M∅+ghosts (14,77±5,87) respect to preM∅ (1,80±1,1) and controls (1,67±0,8) was observed. By gene expression analysis we observed in GD M∅+ghosts an higher HAMP expression (28,13±5,63) compared to preM∅ (2,49±1,5) and controls (0,71±0,02), and a lower SLC40A1 expression (0,01±0,00) compared to GD preM∅ (0,16±0,05) and controls (0,79±0,25). No significant differences in TFRC and GDF11 expression between GD preM∅ and M∅+ghosts in GD patients were observed. These preliminary data suggest that GD macrophages, when stimulated, display a proinflammatory potential. These activated M1 macrophages could contribute to an inflammatory-producing environment, triggering hepcidin and ferroportin expression by an autocrine/paracrine mechanism and leading to dysregulation of iron distribution. Disclosures Cappellini: CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vifor Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Motta:Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees.

A characterization of Gaucher iPS-derived astrocytes: Potential implications for Parkinson's disease

While astrocytes, the most abundant cells found in the brain, have many diverse functions, their role in the lysosomal storage disorder Gaucher disease (GD) has not been explored. GD, resulting from the inherited deficiency of the enzyme glucocerebrosidase and subsequent accumulation of glucosylceramide and its acylated derivative glucosylsphingosine, has both non-neuronopathic (GD1) and neuronopathic forms (GD2 and 3). Furthermore, mutations in GBA1, the gene mutated in GD, are an important risk factor for Parkinson's disease (PD). To elucidate the role of astrocytes in the disease pathogenesis, we generated iAstrocytes from induced pluripotent stem cells made from fibroblasts taken from controls and patients with GD1, with and without PD. We also made iAstrocytes from an infant with GD2, the most severe and progressive form, manifesting in infancy. Gaucher iAstrocytes appropriately showed deficient glucocerebrosidase activity and levels and substrate accumulation. These cells exhibited varying degrees of astrogliosis, Glial Fibrillary Acidic Protein (GFAP) up-regulation and cellular proliferation, depending on the level of residual glucocerebrosidase activity. Glutamte uptake assays demonstrated that the cells were functionally active, although the glutamine transporter EEAT2 was upregulated and EEAT1 downregulated in the GD2 samples. GD2 iAstrocytes were morphologically different, with severe cytoskeletal hypertrophy, overlapping of astrocyte processes, pronounced up-regulation of GFAP and S100β, and significant astrocyte proliferation, recapitulating the neuropathology observed in patients with GD2. Although astrocytes do not express α-synuclein, when the iAstrocytes were co-cultured with dopaminergic neurons generated from the same iPSC lines, excessive α-synuclein released from neurons was endocytosed by astrocytes, translocating into lysosomes. Levels of aggregated α-synuclein increased significantly when cells were treated with monomeric or fibrillar α-synuclein. GD1-PD and GD2 iAstrocytes also exhibited impaired Cathepsin D activity, leading to further α-synuclein accumulation. Cytokine and chemokine profiling of the iAstrocytes demonstrated an inflammatory response. Thus, in patients with GBA1-associated parkinsonism, astrocytes appear to play a role in α-synuclein accumulation and processing, contributing to neuroinflammation.

Screening of intronic mutation IVS9+141A>G in an Indonesian patient with Gaucher disease

Objective: Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by the accumulation of the glycolipid glucosylceramide encoded by the GBA gene in certain organs. At present, more than 460 GBA intronic mutations have been reported in several subpopulations worldwide, but many have never been reported in Indonesia. Here, we aimed to screen for intronic mutations of GBA that might be present in patients with GD in Indonesia. Materials and Methods: Blood samples from patients with and without GD were obtained from the National Dr. Cipto Mangunkusumo Referral Hospital, Jakarta, Indonesia. Genomic DNA samples from peripheral leukocytes were extracted, purified, and amplified using the polymerase chain reaction (PCR) with specific primers. Products of PCR were visualized by gel electrophoresis and were further sequenced to analyze the presence of mutations in intron (intervening sequence [IVS]) 9 of GBA. Results: A mutant allele was identified at IVS9+141A>G, discovered at nucleotide 9335 in IVS 9. This mutation had been reported in India before and was categorized as nonpathogenic. Conclusion: Our study may be used as supplemental information for the GD database in Indonesia and will also open new research opportunities for predicting splicing processes in other intronic variants among patients with GD in Indonesia.

Complement activation causes oncogene expression in Gaucher disease

Abstract GBA1 mutations lead to defective lysosomal glucocerebrosidase resulting in accumulation of glucosylceramide (GC) in Gaucher disease (GD). Patients with GD have an increased risk to develop B cell lymphomas. The exact mechanistic bases for this propensity remain elusive. Recently, we uncovered formation of GC-specific IgG autoantibodies in Gba1 D409V/knockout (Gba19V/−) mice, which recapitulate features of human GD, and in humans with untreated GD. In vivo formation of IgG-GC immune complexes induced massive complement activation and C5a generation. Importantly, C5a-mediated activation of its cognate C5a receptor 1 (C5aR1) on immune cells enhanced GC synthesis, thereby fueling GC accumulation and excess tissue recruitment and activation of inflammatory myeloid and lymphoid immune cells, leading to visceral tissue damage in GD. Here, the expression of Runt-related transcription factor 1 (RUNX-1) was determined in Gba19V/− mice, to evaluate if C5a/C5aR1 axis activation may control the development of lymphomas in GD. RUNX-1 is a member of the Runt oncogene family linked to hematologic malignancies. We determined RUNX-1 expression in tissue from C5aR1 sufficient (+/+) and deficient (−/−) Gba19V/− mice as well as strain-matched control WT and C5aR1−/− mice. Compared to WT, Gba19V/− mice had increased RUNX-1 expression. Strikingly, RUNX-1 expression was markedly downregulated in C5aR−/−Gba19V/− vs. C5aR1+/+Gba19V/− mice. Our findings suggest that the C5a-C5aR1 axis activation in GD drives RUNX1 expression as a novel mechanism to control the development of hematologic malignancies in GD that may be diminished by targeting the C5aR1 axis in GD.

Localization of active endogenous and exogenous β-glucocerebrosidase by correlative light-electron microscopy in human fibroblasts.

β‐Glucocerebrosidase (GBA) is the enzyme that degrades glucosylceramide in lysosomes. Defects in GBA that result in overall loss of enzymatic activity give rise to the lysosomal storage disorder Gaucher disease, which is characterized by the accumulation of glucosylceramide in tissue macrophages. Gaucher disease is currently treated by infusion of mannose receptor‐targeted recombinant GBA. The recombinant GBA is thought to reach the lysosomes of macrophages, based on the impressive clinical response that is observed in Gaucher patients (type 1) receiving this enzyme replacement therapy. In this study, we used cyclophellitol‐derived activity‐based probes (ABPs) with a fluorescent reporter that irreversibly bind to the catalytic pocket of GBA, to visualize the active enzymes in a correlative microscopy approach. The uptake of pre‐labeled recombinant enzyme was monitored by fluorescence and electron microscopy in human fibroblasts that stably expressed the mannose receptor. The endogenous active enzyme was simultaneously visualized by in situ labeling with the ABP containing an orthogonal fluorophore. This method revealed the efficient delivery of recombinant GBA to lysosomal target compartments that contained endogenous active enzyme.

Oxidative Stress Parameters, Trace Elements, and Lipid Profile in Iranian Patients with Gaucher Disease.

Gaucher disease (GD) is most frequent disorder of glycolipid storage. The glucosylceramide accumulation might lead to oxidative stress and changes in lipid profile. Regarding the main role of trace elements in hematopoiesis and oxidative stress, this study was aimed to evaluate the zinc and copper levels, three oxidative stress parameters, and lipid profile in GD. Thirty-three patients with GD along with 64 age- and sex-matched healthy controls participated in the study. The levels of zinc and copper were determined using atomic absorption/flame emission spectrophotometer. Malondialdehyde level was measured using HPLC. Total antioxidant capacity (TAC), catalase activity, and lipid profile were assessed using colorimetric methods. Data were analyzed using SPSS software version 16.0. Significant decrease in the serum levels of Zn (p < 0.001) and Cu (p < 0.001) was observed in patients with GD compared to controls. Subjects in control group showed significantly higher levels of TAC than patients with GD (p < 0.001). In contrast, plasma concentration of malondialdehyde was insignificantly higher in patients with GD than controls (p = 0.06). There was a direct correlation between TAC and hemoglobin concentration (p = 0.035; r = 0.369) in patients with GD. Furthermore, the calculated area under receiver operating characteristic curve for HDL cholesterol was equal to 0.938. The results showed that both zinc and copper levels decreased in patients with GD. Patients with GD showed decreased serum content of TAC. It was found that improving the deficiency of zinc and copper by supplementing them could be useful in management of patients with GD.

GBA haploinsufficiency accelerates alpha-synuclein pathology with altered lipid metabolism in a prodromal model of Parkinson’s disease

Parkinson's disease (PD) is characterized by dopaminergic (DA) cell loss and the accumulation of pathological alpha synuclein (asyn), but its precise pathomechanism remains unclear, and no appropriate animal model has yet been established. Recent studies have shown that a heterozygous mutation of glucocerebrosidase (gba) is one of the most important genetic risk factors in PD. To create mouse model for PD, we crossed asyn Bacterial Artificial Chromosome transgenic mice with gba heterozygous knockout mice. These double-mutant (dm) mice express human asyn in a physiological manner through its native promoter and showed an increase in phosphorylated asyn in the regions vulnerable to PD, such as the olfactory bulb and dorsal motor nucleus of the vagus nerve. Only dm mice showed a significant reduction in DA cells in the substantia nigra pars compacta, suggesting these animals were suitable for a prodromal model of PD. Next, we investigated the in vivo mechanism by which GBA insufficiency accelerates PD pathology, focusing on lipid metabolism. Dm mice showed an increased level of glucosylsphingosine without any noticeable accumulation of glucosylceramide, a direct substrate of GBA. In addition, the overexpression of asyn resulted in decreased GBA activity in mice, while dm mice tended to show an even further decreased level of GBA activity. In conclusion, we created a novel prodromal mouse model to study the disease pathogenesis and develop novel therapeutics for PD and also revealed the mechanism by which heterozygous gba deficiency contributes to PD through abnormal lipid metabolism under conditions of an altered asyn expression in vivo.

Type 1 Gaucher Disease Severity and Risk for Liver Fibrosis in Untreated Patients and in Patients Treated Primarily with Enzyme Replacement Therapy for a Median of 20 Years

Type 1 Gaucher disease (GD1) is a rare, autosomal recessive disorder. Deficient lysosomal glucocerebrosidase activity causes accumulation of glucosylceramide in macrophages that infiltrate bone marrow, spleen and liver. Common clinical manifestations include hematologic cytopenias, hepatosplenomegaly and bone lesions. Since 1991, GD1 has been treated primarily with pharmacological enzyme replacement therapy (ERT). In the pre-ERT era, sequelae of fibrotic liver disease were a common cause of early death. Ultrasonic and MR elastography (MRE) suggest that increased liver stiffness attributed to fibrosis correlates with GD1 disease severity scores. Here, we evaluate the impact of initial and post-ERT (median 20 y, percentiles5-95: 5.2-27.5 y) GD1 severity on probability for liver fibrosis (LF) in 30 patients (pts) using 4 well-established LF prediction scores. We also studied 9 never-treated pts and 7 pts treated for < 18 m and examined the relationship between LF, disease severity scores and D-dimer, ferritin, chitotriosidase and GBA1 genotype.METHODS: 46 adult GD1 pts were evaluated in 2017-18. Pts with known liver disease or on anticoagulation were ineligible. Disease severity was scored with a validated assessment tool GD1-DS3: marked (DS3 6.0-19.0), moderate (DS3 3.0-5.99), mild (DS3 < 3.0). LF probability was assessed with AST/Platelet Ratio Index, Fibrosis-4 Index, Lok Score and NAFLD Fibrosis Score. Scores were classified positive, indeterminate or negative per published standards. Probabilities were stratified as: high (≥ 2 parameters positive), low (≥ 2 parameters negative), or indeterminate (any other score combination).RESULTS: 26/30 pts (86.7%) treated for at least 3 y had low probability of LF at last evaluation. 4/9 (44.4%) ERT-naïve pts and 3/7 (42.9%) pts treated for ≤ 18 m had high probability for LF. 5/7 (71.4%) pts with marked DS3 severity had high LF probability compared to 2/14 (14.3%) with moderate DS3 severity and 3/25 (12.0%) with mild DS3 severity (Table). Of 30 pts treated for ≥ 3 y, 13 had marked DS3 severity scores before starting treatment. Only 1 pt had DS3≥6.00 at the time of last evaluation along with high probability for LF. She had a history of frequent and prolonged treatment interruptions as well as morbid obesity. Of 9 treated pts with moderate baseline severity scores, 4 remained so at last evaluation. One had high probability for LF but also had developed myelodysplasia and severe hemolytic anemia that likely confounded both GD1 severity and LF scores. The third treated pt with high probability for LF had a mild DS3 score but also insulin dependent diabetes mellitus and marked obesity. Chitotriosidase activity was >1000 nMol/mL/h in proportionately more pts with marked disease (5/7, 71.4%), compared to those with moderate (4/14, 28.6%) or mild disease (5/25, 20.0%) and among those pts who were never treated (8/9, 88.9%) or treated for <18 m (4/6, 66.7%), compared to those patients treated for ≥ 3 y. (11/31, 35.5%). All patients who were either untreated or treated for ≤ 18 m had ferritin levels >150 ng/mL, whereas ferritin was increased in less than half the patients treated ≥ 3 y. There was no correlation between these biomarkers and a high probability for LF. No patients had INR> 1.5 or abnormal AST or ALT. D-dimer levels did not correlate with disease severity or probability for LF. Of 10 patients with high probability for LF, 7 had platelet counts < 120x 103/µL. 8/17 (47.1%) with pre-treatment DS3> 6.0 were N370S homozygous compared to 23/28 (82.1%) with baseline moderate or mild severity scores. 7/10 patients with high probability for LF were N370S homozygous.CONCLUSIONS: Per MRE studies reported by others, we find that LF probability based on non-invasive laboratory risk stratification is often increased parallel to GD1-DS3 score. However, in contrast to the pre-ERT era, most adult GD1 patients treated with ERT for a median 20 y, including those with initial marked disease severity, appear unlikely to develop LF. Exceptions may be attributed to confounding concurrent illnesses or to poor treatment compliance. Unlike DS3 score, we found no consistency between traditional GD1 biomarkers and probability for LF. We emphasize that N370S homozygosity can be associated with severe GD1 manifestations including LF and such pts should be treated accordingly. Our assessments of LF probability require verification with elastography especially in pts with thrombocytopenia. [Display omitted] DisclosuresWeinreb:Shire HGT: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genzyme, a Sanofi Company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

The Long-Term Adverse Event Profile of Oral Eliglustat for the Treatment of Gaucher Disease Type 1: Pooled Analysis of Data from 393 Patients in 4 Completed Trials

Background:Gaucher disease type 1 (GD1) is a multi-systemic disorder resulting from deficient activity of the lysosomal enzyme acid β-glucosidase leading to lysosomal accumulation of glucosylceramide (GL-1), primarily in macrophages (Gaucher cells). Thrombocytopenia, anemia, hepatosplenomegaly, and skeletal disease are common presenting symptoms, and hematologists often identify and manage the disease. Eliglustat, an oral substrate reduction therapy, is a first-line treatment for adults with GD1 who have extensive, intermediate, or poor CYP2D6 metabolizer phenotypes (>90% of patients). The clinical trial program for eliglustat is the largest to date for Gaucher disease, and encompasses 2 trials in treatment-naïve patients, 1 trial in stable patients switching to eliglustat after long-term enzyme replacement therapy (the historic standard of care), and a dose regimen study in mostly switch patients.Methods:We analyzed pooled treatment-emergent adverse event (AE) data from all 393 patients with GD1 from 29 countries who received at least 1 eliglustat dose in 4 completed clinical trials sponsored by Sanofi Genzyme: Phase 2 (NCT00358150, N=26), Phase 3 ENGAGE (NCT00891202, N=40), Phase 3 ENCORE (NCT00943111, N=157), and Phase 3 EDGE (NCT01074944, N=170).Results:Mean overall treatment duration in all 393 patients from the 4 completed trials was 3.6 years (maximum: 9.3 years), representing 1400 patient-years of eliglustat exposure. Overall, 81% (n=319) of patients remained in their trial until the availability of commercial eliglustat or study completion. Twenty-five (6%) patients withdrew due to AEs, including 9 patients (2%) with AEs considered related to eliglustat: mild thrombocytopenia (1 patient); mild ventricular tachycardia (1 patient); mild vertigo (1 patient); mild lethargy and mild exfoliative rash (1 patient); mild nausea, mild headache, and moderate anemia (1 patient); moderate arrhythmia (1 patient); moderate palpitations (1 patient); moderate gastroesophageal reflux disease and moderate dyspepsia (1 patient); severe upper abdominal pain (1 patient). Other reasons for study withdrawals were: patient wished to withdraw (n=25, 6%), pregnancy (n=15, 4%), noncompliance (n=3, 1%), and “other” (n=6, 2%). There were 2 on-treatment deaths, neither of which were considered related to eliglustat (downhill skiing accident and cardiac arrest due to hemorrhage after blunt abdominal trauma). Most AEs were mild or moderate (97%) in severity and considered unrelated to eliglustat (86%). Four AEs considered related to eliglustat were reported in ≥5% of patients: dyspepsia (6%), headache (5%), abdominal pain upper (5%), and dizziness (5%). Most of these common related AEs were mild or moderate, occurred only once per patient, and lasted less than 2 weeks. A total of 77 (20%) patients reported at least 1 serious AE, the majority of which were due to hospitalizations for intercurrent illnesses (e.g., appendicitis) and underlying diseases for which Gaucher patients are at increased risk (e.g., femur fracture, joint dislocation, hepatocellular carcinoma, and cholecystitis). Eight patients (2%) had at least 1 serious AE considered related by the investigator: ventricular tachycardia; atrioventricular block and atrioventricular block second degree; peripheral neuropathy; intestinal obstruction; syncope (2 patients); syncope and muscular weakness; arrhythmia. Most were mild or moderate, recovered/resolved, and did not lead to study withdrawal. Most reported cardiac AEs were findings detected in asymptomatic patients during protocol-defined periodic 24‐hour Holter monitoring, were classified as mild, and did not lead to study withdrawal. No clinically significant prolongations of the QTcF interval were observed during extensive electrocardiographic and Holter monitoring in the clinical studies. No differences were observed for the overall frequency of AEs by age group, CYP2D6 metabolizer phenotype, or upper 10th percentile for plasma eliglustat exposure. The proportion of patients reporting AEs decreased over time on eliglustat.Conclusions:This pooled analysis of AE data from one Phase 2 and three Phase 3 completed eliglustat trials demonstrates long-term safety and tolerability of eliglustat in the treatment of adults with GD1. DisclosuresPeterschmitt:Sanofi Genzyme: Employment. Freisens:Sanofi Genzyme: Employment. Hou:Sanofi Genzyme: Employment. Underhill:Sanofi Genzyme: Employment. Foster:Sanofi Genzyme: Employment. Gaemers:Sanofi Genzyme: Employment.

Long-Term Effects of Oral Eliglustat on Skeletal Manifestations of Gaucher Disease Type 1: Results from Four Completed Clinical Trials

Background:In Gaucher disease type 1 (GD1), deficient lysosomal acid β-glucosidase activity leads to accumulation of glucosylceramide and formation of lipid-laden macrophages known as Gaucher cells. Patients experience a range of disease manifestations including anemia, thrombocytopenia, hepatosplenomegaly, and debilitating bone complications that impair quality of life. Hematologists are central to disease recognition and management. Most patients, even those without overt symptoms of bone involvement, have radiologic evidence of bone disease. Eliglustat, an oral substrate-reduction therapy, is a first-line treatment for adults with GD1 who have extensive, intermediate or poor CYP2D6 metabolizer phenotypes (>90% of patients). Oral eliglustat partially inhibits glucosylceramide synthase, thereby reducing glucosylceramide accumulation, unlike enzyme replacement therapy (ERT), the historic standard of care for GD1, which augments acid β-glucosidase activity in mononuclear phagocytes and is administered in biweekly infusions.Methods:We analyzed bone mineral density T- and Z-scores and occurrence of bone pain and bone crises in 4 completed clinical trials sponsored by Sanofi Genzyme that involved 393 GD1 patients treated with eliglustat for 4-8 years: Phase 2 (NCT00358150), Phase 3 ENGAGE (NCT00891202), Phase 3 ENCORE (NCT00943111), and Phase 3 EDGE (NCT01074944). Healthy reference scores are >-1 for T-scores and >-2 for Z-scores. Bone marrow burden (BMB) scores and changes in macrophage inflammatory protein 1β (MIP-1β) were also analyzed in the 3 Phase 3 trials. BMB, an MRI-based scoring system to evaluate marrow infiltration of both the lumbar spine and femur, and MIP-1β, a biomarker produced by inflammatory phagocytes surrounding Gaucher cells, are associated with active and evolving bone disease in GD1.Results:In treatment-naïve patients (Phase 2, N=26; ENGAGE, N=40), mean spine T-scores moved from the osteopenic range (>-2.5 to ≤ -1) at baseline to the healthy reference range after 2-3 years of eliglustat, with continued improvement thereafter in both trials. Mean±SEM spine T-score increased from -1.55±0.28 to -0.59±0.34 (n=14; mean±SEM increase from baseline: 0.96±0.32), after 8 years in Phase 2, and increased to -0.53±0.27 in ENGAGE patients with bone data at 4.5 years (n=9; mean±SEM increase from baseline: 0.53±0.16). Spine Z-scores for both trials showed similar improvements; femur T- and Z-scores in both trails remained in the normal range. In the ENCORE trial of patients stabilized after a mean of 10 years of ERT (N=157), T- and Z-scores (spine and femur) remained in the reference ranges for up to 4 years; least square mean spine Z-scores improved by 0.29 (P<0.0001). In EDGE (N=170, mostly ERT switch), randomized patients stabilized on eliglustat during a 6- to 18-month Lead-In Period maintained normal T- and Z-scores (spine and femur) through the dose regimen arm of the study and the succeeding extension period. Mean total BMB scores improved from 10 (in the marked to severe range) to 8 (moderate range) in ENGAGE (treatment-naive patients), and remained stable at 8 throughout ENCORE and EDGE (switch/mostly switch patients). The proportion of patients experiencing recent (≤4 weeks) bone pain decreased after treatment in all 4 trials, and reported pain became less severe. No patient in Phase 2 or ENGAGE had a bone crisis while on eliglustat. Bone crises were reported in 3/157 (1.9%) ENCORE patients over the trial duration. In EDGE, the only trial to enroll patients with active bone disease, 6/170 (3.5%) patients had bone crises during the trial, 3 of whom had bone crises during the 6 months prior to trial entry. Bone crises decreased with increasing time on eliglustat, with only 1 bone crisis reported during the 2- to 3-year trial extension. In ENGAGE (treatment-naïve), median MIP-1β levels elevated 3-fold at baseline normalized. In ENCORE and EDGE (switch/mostly switch), MIP-1β levels remained in the normal range. Across the 4 trials, most adverse events were mild/moderate (97%) and considered unrelated to eliglustat (86%); 9 (2.3% overall) patients withdrew due to adverse events considered drug-related.Conclusions:Long-term eliglustat treatment induced continued improvement in bone parameters in treatment-naïve patients. These parameters remained stable in trials involving patients who had been previously treated. Eliglustat was generally well-tolerated. DisclosuresMistry:Sanofi Genzyme: Honoraria, Other: Principal Investigator for the eliglustat ENGAGE and ENCORE trials; ReceivedTravel Reimbursement; Member of the ICCG North American Advisory Board; , Research Funding. Charrow:Sanofi Genzyme: Honoraria, Other: Principal investigator, eliglustat EDGE trial; Member of advisory boards, Research Funding. Cox:Sanofi Genzyme: Honoraria, Other: Principal investigator for the eliglustat ENCORE trial; received travel reimbursement, Research Funding. Lukina:Sanofi Genzyme: Honoraria, Other: Principal investigator in the eliglustat Phase 2, ENGAGE, ENCORE, and EDGE trials; receives travel reimbursement; member of advisory boards, Research Funding. Marinakis:Sanofi Genzyme: Other: Principal investigator for the eliglustat EDGE trial, Research Funding. Foster:Sanofi Genzyme: Employment. Gaemers:Sanofi Genzyme: Employment. Peterschmitt:Sanofi Genzyme: Employment.

Glucocerebrosidase deficiency promotes protein aggregation through dysregulation of extracellular vesicles.

Mutations in the glucosylceramidase beta (GBA) gene are strongly associated with neurodegenerative diseases marked by protein aggregation. GBA encodes the lysosomal enzyme glucocerebrosidase, which breaks down glucosylceramide. A common explanation for the link between GBA mutations and protein aggregation is that lysosomal accumulation of glucosylceramide causes impaired autophagy. We tested this hypothesis directly by measuring protein turnover and abundance in Drosophila mutants with deletions in the GBA ortholog Gba1b. Proteomic analyses revealed that known autophagy substrates, which had severely impaired turnover in autophagy-deficient Atg7 mutants, showed little to no overall slowing of turnover or increase in abundance in Gba1b mutants. Likewise, Gba1b mutants did not have the marked impairment of mitochondrial protein turnover seen in mitophagy-deficient parkin mutants. Proteasome activity, microautophagy, and endocytic degradation also appeared unaffected in Gba1b mutants. However, we found striking changes in the turnover and abundance of proteins associated with extracellular vesicles (EVs), which have been proposed as vehicles for the spread of protein aggregates in neurodegenerative disease. These changes were specific to Gba1b mutants and did not represent an acceleration of normal aging. Western blotting of isolated EVs confirmed the increased abundance of EV proteins in Gba1b mutants, and nanoparticle tracking analysis revealed that Gba1b mutants had six times as many EVs as controls. Genetic perturbations of EV production in Gba1b mutants suppressed protein aggregation, demonstrating that the increase in EV abundance contributed to the accumulation of protein aggregates. Together, our findings indicate that glucocerebrosidase deficiency causes pathogenic changes in EV metabolism and may promote the spread of protein aggregates through extracellular vesicles.

Quantitative analysis of recombinant glucocerebrosidase brain delivery via lipid nanoparticles

Gaucher disease (GD), the most prevalent genetic lysosomal storage disease, is characterized by the accumulation of glucosylceramide, mainly in monocyte-derived cells, due to deficient activity of lysosomal acid-β-glucocerebrosidase (GCase). The disease is heterogeneous and may vary from a very mild visceral disease to a severe neuronopathic disease, with very early death during the first years of life. Two therapeutic modalities are in use today; enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). Neither of the two modalities are applicable for patients with the neuronopathic forms of GD. While the infused enzyme in ERT cannot cross the blood–brain-barrier, SRT is not suitable for young patients. Herein, we investigated novel approaches to deliver recombinant GCase (rGCase) into the brain using lipid nanoparticles (LNPs). These LNPs were composed of a mixture of negative, positive and zwitterion phospholipids and were delivered intranasally into the brains of mice. A quantitative analysis performed intranasally in mice revealed a dramatic accumulation of the enzyme that was formulated into the LNPs in the brains of the mice (3.91% ± 0.3% injected dose (ID)/mg tissue)) versus the free enzyme (0.29% ± 0.07, % ID/mg tissue). The administrated particle-delivered enzymes were able to enter the brain parenchyma and accumulate in the CD11b+ cells, which are the target cells in GD. When supplied to GD-derived skin fibroblasts, a 35% ± 1.2 increase in intracellular GCase activity was measured only with the LNP-encapsulated enzyme. This strategy may pave the way for novel therapeutic modalities to treat GD and other diseases such as Alzheimer’s and Parkinson’s.

Fludarabine-resistance associates with ceramide metabolism and leukemia stem cell development in chronic lymphocytic leukemia.

Fludarabine (flu) -containing regimens such as flu, cyclophosphamide and rituximab have been established as one of the standard first line therapy in medically-fit chronic lymphocytic leukemia (CLL) patients. Therefore, flu-refractory (primary flu-insensitivity or flu-caused relapse) remains a major problem causing treatment failure for CLL patients. We isolated the peripheral blood mononuclear cells (PBMCs) from CLL patients and treated with flu to find flu-refractory cases, and established flu-resistant clonal cells to study molecular mechanism of flu-resistance. By comparing parental MEC-2 cells, a human CLL cell line, we found that flu-resistant clonal cells were significantly increased lethal dose 50 of flu concentration, and up-regulated expression of P-glycoprotein, a drug-resistant marker, glucosylceramide synthase (GCS), an enzyme that can convert ceramide to glucosylceramide, and CD34, a leukemia stem cell marker. Overexpression of GCS leads to promptly elimination of cellular ceramide levels and accumulation of glucosylceramide, which reduces apoptosis and promotes survival and proliferation of flu-resistant clonal cells. Furthermore, we demonstrated that the accumulation of glucosylceramide can be blocked by PDMP to restore flu-sensitivity in flu-resistant clonal cells. We also found that elevating glucosylceramide levels in flu-resistant clonal cells was associated with up-regulation of GCS and CD34 expression. Importantly, overexpression of GCS or CD34 was also determined in flu-refractory PBMCs. Our results show that flu-resistance is associated with the alteration of ceramide metabolism and the development of leukemia stem cell-like cells. The flu-resistance can be reversed by GCS inhibition as a novel strategy for overcoming drug resistance.

Generation of osteoclasts from type 1 Gaucher patients and correlation with clinical and genetic features of disease

Gaucher disease (GD) is a rare autosomal recessive disorder caused by deficient activity of β-glucocerebrosidase resulting in the accumulation of glucosylceramide. Bone disease is a common feature with radiological evidence in up to 93% of patients. Severity of bone involvement ranges from osteoporosis to pathological fractures. The progressive course of type 1 GD is largely mitigated by treatment with enzyme replacement therapy (ERT) or substrate reduction. A number of studies have shown some patients suffer bone events while receiving ERT. Studies of biochemical markers of bone turnover have generated varied results and as a consequence are not generally used to assess bone disease in GD. In vitro osteoclast generation from peripheral blood samples of 74 Gaucher patients followed over a period of up to 10 years was correlated with bone events, reports of bone pain, anaemia, spleen status, bone mineral density, chitotriosidase activity, treatment with Gaucher specific therapies, bisphosphonates, mutation status and severity. Osteoclast generation, enumerated when cultured on glass, was significantly higher when differentiated from the peripheral blood of Gaucher patients which reported bone pain (116.4 ± 18.0 vs 69.0 ± 8.6, p < 0.01), had anaemia (153.7 ± 34.9 vs 78.5 ± 8.8, p < 0.01), had a splenectomy (137.6 ± 41.1 vs 60.8 ± 13.0, p < 0.05), versus those who did not. Osteoclast generation was also indicative of in vivo Gaucher specific therapy response as those naïve to therapy generated significantly more osteoclasts than those on therapy (111.2 ± 35.8 vs 45.1 ± 10.3, p < 0.05), as did patients receiving therapy but still suffering bone events (125.1 ± 31.37 vs 45.1 ± 10.33, p < 0.05). These findings demonstrate that the in vitro osteoclast assay may be a useful method for following bone disease progression in Gaucher patients.

Absence of infiltrating peripheral myeloid cells in the brains of mouse models of lysosomal storage disorders

Approximately 70 lysosomal storage diseases are currently known, resulting from mutations in genes encoding lysosomal enzymes and membrane proteins. Defects in lysosomal enzymes that hydrolyze sphingolipids have been relatively well studied. Gaucher disease is caused by the loss of activity of glucocerebrosidase, leading to accumulation of glucosylceramide. Gaucher disease exhibits a number of subtypes, with types 2 and 3 showing significant neuropathology. Sandhoff disease results from the defective activity of β-hexosaminidase, leading to accumulation of ganglioside GM2. Niemann-Pick type C disease is primarily caused by the loss of activity of the lysosomal membrane protein, NPC1, leading to storage of cholesterol and sphingosine. All three disorders display significant neuropathology, accompanied by neuroinflammation. It is commonly assumed that neuroinflammation is the result of infiltration of monocyte-derived macrophages into the brain; for instance, cells resembling lipid-engorged macrophages ('Gaucher cells') have been observed in the brain of Gaucher disease patients. We now review the evidence that inflammatory macrophages are recruited into the brain in these diseases and then go on to provide some experimental data that, at least in the three mouse models tested, monocyte-derived macrophages do not appear to infiltrate the brain. Resident microglia, which are phenotypically distinct from infiltrating macrophages, are the only myeloid population present in significant numbers within the brain parenchyma in these authentic mouse models, even during the late symptomatic stages of disease when there is substantial neuroinflammation. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. This article is part of the Special Issue "Lysosomal Storage Disorders".

NEW INSIGHTS INTO RARE METABOLIC DISEASES IN PAEDIATRIC PRACTICE

Rare diseases are those that affect at most one in 2000 persons worldwide. According to the date around 6000-7000 rare diseases have been described until now. Although there are so many differences between rare diseases patents that are affected are facing with the more or less same difficulties. Before a final diagnose they usually spend a few years losing a precious time for the treatment. The aim of this review article is to give insight into a several rare diseases that are successfully treated by enzyme replacement therapy (ERT). A complete search of the Cochrane Central Register of Controlled Trials (CENTRAL) in the Cochrane Library, MEDLINE and Pub Med up to January 2018 was carried out. Fabry disease (FD) is a progressive, X-linked inherited disorder of glycosphingolipid metabolism due to deficient or absent lysosomal a-galactosidase A activity. Gaucher disease is characterized by a deficiency of acid β-glucosidase and accumulation of glucosylceramide within lysosomes of tissue macrophages. The accumulation of glucosylceramide is responsible for the manifestations of Gaucher disease, including hepatomegaly, splenomegaly, thrombocytopenia, and anemia. MPS I characterized the defect in the gene for α-L-iduronidase (IUDA), an enzyme that degrades glycosaminoglycans (GAGs) in lysosomes. Pompe disease is a rare, progressive, debilitating and often fatal neuromuscular disorder resulting from the deficiency of a lysosomal enzyme, acid alpha-glucosidase (GAA). Enzyme testing on Dried Blood Spots (DBS) is a 1st tier/screening test for all four above mentioned metabolic rare diseases. An enzyme replacement therapy is the only effective treatment patients with Fabry disease, Pompe disease, Gausher disease and MPS I.

Reduction of lipid accumulation rescues Bietti’s crystalline dystrophy phenotypes

Bietti's crystalline dystrophy (BCD) is an intractable and progressive chorioretinal degenerative disease caused by mutations in the CYP4V2 gene, resulting in blindness in most patients. Although we and others have shown that retinal pigment epithelium (RPE) cells are primarily impaired in patients with BCD, the underlying mechanisms of RPE cell damage are still unclear because we lack access to appropriate disease models and to lesion-affected cells from patients with BCD. Here, we generated human RPE cells from induced pluripotent stem cells (iPSCs) derived from patients with BCD carrying a CYP4V2 mutation and successfully established an in vitro model of BCD, i.e., BCD patient-specific iPSC-RPE cells. In this model, RPE cells showed degenerative changes of vacuolated cytoplasm similar to those in postmortem specimens from patients with BCD. BCD iPSC-RPE cells exhibited lysosomal dysfunction and impairment of autophagy flux, followed by cell death. Lipidomic analyses revealed the accumulation of glucosylceramide and free cholesterol in BCD-affected cells. Notably, we found that reducing free cholesterol by cyclodextrins or δ-tocopherol in RPE cells rescued BCD phenotypes, whereas glucosylceramide reduction did not affect the BCD phenotype. Our data provide evidence that reducing intracellular free cholesterol may have therapeutic efficacy in patients with BCD.