Increased glucosylsphingosine levels and Gaucher disease in GBA1-associated Parkinson's disease.

INTRODUCTION: Gaucher's disease (GD) is caused by biallelic mutations in the GBA1 gene, leading to reduced glucocerebrosidase (GCase) activity and substrate (glucosylceramide and glucosylsphingosine, GlcSph) accumulation. GBA1 variant carriers are at risk of Parkinson's disease (PD), but only those with biallelic mutations cross the threshold of GCase reduction, leading to substrate accumulation and GD. The link between GBA1 mutations, GD and PD is not fully understood. Here we aimed at reporting the results of a large PD population screening with dried blood spot tests for GD. METHODS: We measured GCase activity and GlcSph levels in 1344 PD patients with dried blood spot tests, and performed GBA1 genetic sequencing. RESULTS: While the GCase activity was reduced in GBA1-PD carriers compared to wild type PD, GlcSph was increased in GBA1-PD compared to GBA1-controls, regardless of the underlying type of GBA1 variant. 13.6 % and 0.4 % of PD patients had mono- or biallelic GBA1 mutations respectively. GCase deficiency, lipid accumulation and clinical manifestations of GD was detected in five PD patients with biallelic GBA1 mutations, of whom four had a risk combined with a GD causing variant. CONCLUSIONS: GlcSph appearing higher in PD may represent a reliable biomarker of the disease and deserves to be further investigated. This study highlights the importance of screening PD patients for possible underlying GD, which is a treatable condition that should not be missed. We diagnosed GD cases carrying a "risk" variant in one allele, which is an unprecedented finding deserving further investigation.

Promyelinating drugs ameliorate oligodendrocyte pathologies in a mouse model of Krabbe disease.

Krabbe disease (KD) is a rare inherited demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramide (GalCer) ß-galactosidase. Most patients with KD exhibit fatal cerebral demyelination with apoptotic oligodendrocyte (OL) death and die before the age of 2-4 years. We have previously reported that primary OLs isolated from the brains of twitcher (twi) mice, an authentic mouse model of KD, have cell-autonomous developmental defects and undergo apoptotic death accompanied by abnormal accumulation of psychosine, an endogenous cytotoxic lyso-derivative of GalCer. In this study, we aimed to investigate the effects of the preclinical promyelinating drugs clemastine and Sob-AM2 on KD OL pathologies using primary OLs isolated from the brains of twi mice. Both agents specifically prevented the apoptotic death observed in twi OLs. However, while Sob-AM2 showed higher efficacy in restoring the impaired differentiation and maturation of twi OLs, clemastine more potently reduced the endogenous psychosine levels. These results present the first preclinical in vitro data, suggesting that clemastine and Sob-AM2 can act directly and distinctly on OLs in KD and ameliorate their cellular pathologies associated with myelin degeneration.

A Brazilian Rare-Disease Center's Experience with Glucosylsphingosine (lyso-Gb1) in Patients with Gaucher Disease: Exploring a Novel Correlation with IgG Levels in Plasma and a Biomarker Measurement in CSF.

Gaucher disease (GD, OMIM 230800) is one of the most common lysosomal disorders, being caused by the deficient activity of the enzyme acid ß-glucocerebrosidase (Gcase). Three clinical forms of Gaucher's disease (GD) are classified based on neurological involvement. Type 1 (GD1) is non-neuronopathic, while types 2 (GD2) and 3 (GD3) are neuronopathic forms. Gcase catalyzes the conversion of glucosylceramide (GlcCer) into ceramide and glucose. As GlcCer accumulates in lysosomal macrophages, it undergoes deacylation to become glycosylsphingosine (lyso-Gb1), which has shown to be a useful and reliable biomarker for the diagnosis and monitoring of treated and untreated patients with GD. Multiple myeloma (MM) is one of the leading causes of cancer-related death among patients with GD and monoclonal gammopathy of undetermined significance (MGUS) is a non-neoplastic condition that can be a telltale sign of a B clonal proliferation caused by the chronic activation of B cells. This study aimed to quantify Lyso-Gb1 levels in dried blood spots (DBS) and cerebrospinal fluid (CSF) as biomarkers for Gaucher disease (GD) and discuss the association of this biomarker with other clinical parameters. This is a mixed-methods study incorporating both cross-sectional and longitudinal elements within a cohort design with a convenience-sampling strategy. Data collection took place from January 2012 to March 2023. Lyso-Gb1 extraction from DBS involved the use of a methanol-acetonitrile-water mixture, followed by incubation and centrifugation. Analysis was performed using UPLC-MS/MS with MassLynx software version 4.2 and the control group for the DBS measurements included general newborns. CSF Lyso-Gb1 was extracted using ethyl acetate, analyzed by UPLC-MS/MS with a calibration curve, and expressed in pmol/L. Lysosomal activity in CSF was assessed by measuring chitotriosidase (Cht), and other lysosomal enzyme activities were assessed as previously described in the literature. Patients with metachromatic leukodystrophy (MLD) were used as controls. Thirty-two treated patients (twenty-nine GD1 and three GD3, all on ERT except for one GD type on SRT with eliglustat) and three untreated patients (one GD1, one GD2, and one GD3) were included. When analyzing only the treated GD1 group, a significant correlation was found between lyso-Gb1 and age (rho = -0.447, p = 0.001), ChT, and IgG levels (rho = 0.73, p < 0.001; and rho = 0.36, p = 0.03, respectively). Five GD1 patients (three females, mean age 40 years) also had their CSF collected and analyzed. The average measurement of lyso-Gb1 in CSF was 94 pmol/L (range: 57.1-157.9 pmol/L) versus <6.2 pmol/L in the control group (MLD). This is the first time, to the best of our knowledge, that lyso-Gb1 has been associated with IgG levels. While this finding reflects a risk for MGUS or MM and not only chronic plasma B-cell activation, it still requires further studies. Moreover, the analysis of CSF lyso-Gb1 levels in GD1 patients was demonstrated to be significantly higher than the control group. This raises the hypothesis that CSF lyso-Gb1 may serve as a valuable indicator for neurological involvement in GD, providing insights into the potential implications for neurological manifestations in GD, including GD1. The correlation between lyso-Gb1 and ChT levels in treated GD1 patients further underscores the interconnectedness of lysosomal markers and their relevance in monitoring.

A Comparative Biochemical and Pathological Evaluation of Brain Samples from Knock-In Murine Models of Gaucher Disease.

Gaucher disease (GD) is a lysosomal storage disorder stemming from biallelic mutations in GBA1, characterized by glucocerebrosidase dysfunction and glucocerebroside and glucosylsphingosine accumulation. Since phenotypes of murine models of GD often differ from those in patients, the careful characterization of Gba1 mutant mice is necessary to establish their ability to model GD. We performed side-by-side comparative biochemical and pathologic analyses of four murine Gba1 models with genotypes L444P/L444P (p.L483P/p.L483P), L444P/null, D409H/D409H (p.D448H/p.D448H) and D409H/null, along with matched wildtype mice, all with the same genetic background and cage conditions. All mutant mice exhibited significantly lower glucocerebrosidase activity (p < 0.0001) and higher glucosylsphingosine levels than wildtype, with the lowest glucocerebrosidase and the highest glucosylsphingosine levels in mice carrying a null allele. Although glucocerebrosidase activity in L444P and D409H mice was similar, D409H mice showed more lipid accumulation. No Gaucher or storage-like cells were detected in any of the Gba1 mutant mice. Quantification of neuroinflammation, dopaminergic neuronal loss, alpha-synuclein levels and motor behavior revealed no significant findings, even in aged animals. Thus, while the models may have utility for testing the effect of different therapies on enzymatic activity, they did not recapitulate the pathological phenotype of patients with GD, and better models are needed.

Effects of sulfatide on peripheral nerves in metachromatic leukodystrophy.

OBJECTIVE: To evaluate the longitudinal correlations between sulfatide/lysosulfatide levels and central and peripheral nervous system function in children with metachromatic leukodystrophy (MLD) and to explore the impact of intravenous recombinant human arylsulfatase A (rhASA) treatment on myelin turnover. METHODS: A Phase 1/2 study of intravenous rhASA investigated cerebrospinal fluid (CSF) and sural nerve sulfatide levels, 88-item Gross Motor Function Measure (GMFM-88) total score, sensory and motor nerve conduction, brain N-acetylaspartate (NAA) levels, and sural nerve histology in 13 children with MLD. Myelinated and unmyelinated nerves from an untreated MLD mouse model were also analyzed. RESULTS: CSF sulfatide levels correlated with neither Z-scores for GMFM-88 nor brain NAA levels; however, CSF sulfatide levels correlated negatively with Z-scores of nerve conduction parameters, number of large (≥7 µm) myelinated fibers, and myelin/fiber diameter slope, and positively with nerve g-ratios and cortical latencies of somatosensory-evoked potentials. Quantity of endoneural litter positively correlated with sural nerve sulfatide/lysosulfatide levels. CSF sulfatide levels decreased with continuous high-dose treatment; this change correlated with improved nerve conduction. At 26 weeks after treatment, nerve g-ratio decreased by 2%, and inclusion bodies per Schwann cell unit increased by 55%. In mice, abnormal sulfatide storage was observed in non-myelinating Schwann cells in Remak bundles of sciatic nerves but not in unmyelinated urethral nerves. INTERPRETATION: Lower sulfatide levels in the CSF and peripheral nerves correlate with better peripheral nerve function in children with MLD; intravenous rhASA treatment may reduce CSF sulfatide levels and enhance sulfatide/lysosulfatide processing and remyelination in peripheral nerves.

Activation of serotonin receptor 2 by glucosylsphingosine can be enhanced by TRPA1 but not TRPV1: Implication of a novel glucosylsphingosine-mediated itch pathway.

Glucosylsphingosine (GS) is an endogenous sphingolipid that specifically accumulates in the skin of patients with atopic dermatitis (AD). Notably, it was recently found that GS can induce itch sensation by activating serotonin receptor 2A and TRPV4 ion channels. However, it is still uncertain whether other molecules are involved in GS-induced itch sensation. Therefore, by using the calcium imaging technique, we investigated whether serotonin receptor 2 - specifically 2A and 2B - can interact with TRPV1 and TRPA1, because these are representative ion channels in the transmission of itch. As a result, it was found that GS did not activate TRPV1 or TRPA1 per se. Moreover, cells expressing both serotonin receptor 2 and TRPV1 did not show any changes in calcium responses. However, enhanced calcium responses were observed in cells expressing serotonin receptor 2 and TRPA1, suggesting a possible interaction between these two molecules. Similar synergistic effects were also observed in cells expressing serotonin receptor 2 and TRPA1, but not TRPV1. Furthermore, a phospholipase C inhibitor (U73122) and a store-operated calcium entry blocker (SKF96365) significantly reduced GS-induced responses in cells expressing both serotonin receptor 2 and TRPA1, but not with pre-treatment with a Gßγ-complex blocker (gallein). Therefore, we propose a putative novel pathway for GS-induced itch sensation, such that serotonin receptor 2 could be coupled to TRPA1 but not TRPV1 in sensory neurons.

Highly-sensitive simultaneous quantitation of glucosylsphingosine and galactosylsphingosine in human cerebrospinal fluid by liquid chromatography/tandem mass spectrometry.

Mutations in the GBA gene, encoding glucocerebrosidase (GCase), are linked to Gaucher disease (GD) and are the most common risk factors for Parkinson's disease (PD). The glucosylsphingosine (GlcSph) in cerebrospinal fluid (CSF) is used as a pharmacodynamic marker for GCase functionalizing therapy in GD patients. Its isobaric structural isomer, galactosylsphingosine (GalSph, psychosine), is also used as a diagnostic blood marker in Krabbe disease (KD) which is caused by a deficiency in ß-galactocerebrosidase (GALC). However, there are no reports of GlcSph quantification in the CSF of GBA-PD patients and normal healthy humans due to low concentrations. In this study, we successfully quantified GlcSph in healthy human CSF using a highly sensitive LC-MS/MS method with separation of GalSph. The lower limit of quantitation (LLOQ) was 0.1 pg/mL. Additionally, GlcSph and GalSph concentrations in the plasma and brain were determined using different LC-MS/MS methods. The mean concentrations of GlcSph and GalSph in normal human CSF were 1.07 and 9.44 pg/mL, respectively. The GalSph level in the CSF and brain was higher than that of GlcSph, whereas plasma GalSph was lower than GlcSph. Because GCase and GALC are expressed in the brain and the peripheral tissues, GlcSph and GalSph in CSF would be a good surrogate of concentration change in the brain by targeted therapies. This method measures normal levels of GlcSph and GalSph in healthy human CSF without accumulation of sphingolipids, and confirms whether abnormal CSF concentrations can be reduced to normal levels by therapy.

Consequences of excessive glucosylsphingosine in glucocerebrosidase-deficient zebrafish.

In 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.

Gaucher Disease Diagnosis Using Lyso-Gb1 on Dry Blood Spot Samples: Time to Change the Paradigm?

For years, the gold standard for diagnosing Gaucher disease (GD) has been detecting reduced ß-glucocerebrosidase (GCase) activity in peripheral blood cells combined with GBA1 mutation analysis. The use of dried blood spot (DBS) specimens offers many advantages, including easy collection, the need for a small amount of blood, and simpler transportation. However, DBS has limitations for measuring GCase activity. In this paper, we recount our cross-sectional study and publish seven years of experience using DBS samples and levels of the deacylated form of glucocerebroside, glucosylsphingosine (lyso-Gb1), for GD diagnosis. Of 444 screened subjects, 99 (22.3%) were diagnosed with GD at a median (range) age of 21 (1-78) years. Lyso-Gb levels for genetically confirmed GD patients vs. subjects negative to GD diagnosis were 252 (9-1340) ng/mL and 5.4 (1.5-16) ng/mL, respectively. Patients diagnosed with GD1 and mild GBA1 variants had lower median (range) lyso-Gb1, 194 (9-1050), compared to GD1 and severe GBA1 variants, 447 (38-1340) ng/mL, and neuronopathic GD, 325 (116-1270) ng/mL (p = 0.001). Subjects with heterozygous GBA1 variants (carrier) had higher lyso-Gb1 levels, 5.8 (2.5-15.3) ng/mL, compared to wild-type GBA1, 4.9 (1.5-16), ng/mL (p = 0.001). Lyso-Gb1 levels, median (range), were 5 (2.7-10.7) in heterozygous GBA1 carriers with Parkinson's disease (PD), similar to lyso-Gb1 levels in subjects without PD. We call for a paradigm change for the diagnosis of GD based on lyso-Gb1 measurements and confirmatory GBA1 mutation analyses in DBS. Lyso-Gb1 levels could not be used to differentiate between heterozygous GBA1 carriers and wild type.

Abdominal lymphadenopathy in children with Gaucher disease: Relation to disease severity and glucosylsphingosine.

Few case reports and series reported abdominal lymphadenopathy (ALN) in people with Gaucher disease (GD). However, it's prevalence among Gaucher population, clinical implications and potential biomarkers are unknown. Hence this study aims to assess the prevalence of ALN among children with GD & to correlate it to neutrophil-lymphocytic-ratio (NLR), platelet-lymphocytic-ratio (PLR) and glucosylsphingosine (Lyso-GL1). Fifty children with GD (14 type-1 and 36 type-3) on enzyme-replacement therapy (ERT) were compared to 50 matched healthy controls, focusing on history of pressure manifestations by ALN (diarrhea, constipation, abdominal pain, intestinal obstruction), and history of splenectomy, with calculation of severity scoring index (SSI). NLR, PLR and Lyso-GL1 were measured. Abdominal-ultrasound was done with assessment of liver and spleen volumes and ALN. CT-scan was done for those having significant lymphadenopathy. Twenty-six children with GD had ALN (52%). The most common presentations were abdominal-pain (22%) & constipation (18%), with intestinal-obstruction in 3 children (6%). Children with GD had significantly higher NLR (p < .001) and decreased PLR (p = .024) compared to controls. Interestingly, children with GD having ALN had significantly higher SSI (.012), Lyso-GL1 (p = .002) and NLR (p = .001) than those without ALN. Multivariate-logistic regression showed that ALN was independently related to Lyso-GL1 (p = .027), NLR (p = .023) and SSI (p = .032). Thus, ALN is a prevalent GD morbidity with wide clinical-spectrum ranging from asymptomatic cases to intestinal obstruction. ALN is related to SSI, NLR and Lyso-GL1 in children with GD.HighlightsChildren with GD had significantly higher NLR and lower PLR compared to controls.Children with GD having ALN had significantly higher SSI, Lyso-GL1 and NLR than those without ALN.ALN was independently related to Lyso-GL1, NLR and SSI in children with GD.

Plasma Glucosylsphingosine in GBA1 Mutation Carriers with and without Parkinson's Disease.

BACKGROUND: Biallelic mutations in the GBA1 gene encoding glucocerebrosidase cause Gaucher's disease, whereas heterozygous carriers are at risk for Parkinson's disease (PD). Glucosylsphingosine is a clinically meaningful biomarker of Gaucher's disease but could not be assayed previously in heterozygous GBA1 carriers. OBJECTIVE: The aim of this study was to assess plasma glucosylsphingosine levels in GBA1 N370S carriers with and without PD. METHODS: Glucosylsphingosine, glucosylceramide, and four other lipids were quantified in plasma from N370S heterozygotes with (n = 20) or without (n = 20) PD, healthy controls (n = 20), idiopathic PD (n = 20), and four N370S homozygotes (positive controls; Gaucher's/PD) using quantitative ultra-performance liquid chromatography tandem mass spectrometry. RESULTS: Plasma glucosylsphingosine was significantly higher in N370S heterozygotes compared with noncarriers, independent of disease status. As expected, Gaucher's/PD cases showed increases in both glucocerebrosidase substrates, glucosylsphingosine and glucosylceramide. CONCLUSIONS: Plasma glucosylsphingosine accumulation in N370S heterozygotes shown in this study opens up its future assessment as a clinically meaningful biomarker of GBA1-PD. © 2021 International Parkinson and Movement Disorder Society.

Glucosylsphingosine evokes pruritus via activation of 5-HT2A receptor and TRPV4 in sensory neurons.

BACKGROUND AND PURPOSE: Glucosylsphingosine (GS), an endogenous sphingolipid, is highly accumulated in the epidermis of patients with atopic dermatitis (AD) due to abnormal ceramide metabolism. More importantly, GS can evoke scratching behaviours. However, the precise molecular mechanism by which GS induces pruritus has been elusive. Thus, the present study aimed to elucidate the molecular signalling pathway of GS, especially at the peripheral sensory neuronal levels. EXPERIMENTAL APPROACH: Calcium imaging was used to investigate the responses of HEK293T cells or mouse dorsal root ganglion (DRG) neurons to application of GS. Scratching behaviour tests were also performed with wild-type and Trpv4 knockout mice. KEY RESULTS: GS activated DRG neurons in a manner involving both the 5-HT2A receptor and TRPV4. Furthermore, GS-induced responses were significantly suppressed by various inhibitors, including ketanserin (5-HT2A receptor antagonist), YM254890 (Gαq/11 inhibitor), gallein (Gßγ complex inhibitor), U73122 (phospholipase C inhibitor), bisindolylmaleimide I (PKC inhibitor) and HC067047 (TRPV4 antagonist). Moreover, DRG neurons from Trpv4 knockout mice exhibited significantly reduced responses to GS. Additionally, GS-evoked scratching behaviours were greatly decreased by pretreatment with inhibitors of either 5-HT2A receptor or TRPV4. As expected, GS-evoked scratching behaviour was also significantly decreased in Trpv4 knockout mice. CONCLUSION AND IMPLICATIONS: Overall, the present study provides evidence for a novel molecular signalling pathway for GS-evoked pruritus, which utilizes both 5-HT2A receptor and TRPV4 in mouse sensory neurons. Considering the high accumulation of GS in the epidermis of patients with AD, GS could be another pruritogen in patients with AD.

Deletion of fatty acid amide hydrolase reduces lyso-sulfatide levels but exacerbates metachromatic leukodystrophy in mice.

An inherited deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) characterized by massive intralysosomal storage of the acidic glycosphingolipid sulfatide and progressive demyelination. Lyso-sulfatide, which differs from sulfatide by the lack of the N-linked fatty acid, also accumulates in MLD and is considered a key driver of pathology although its concentrations are far below sulfatide levels. However, the metabolic origin of lyso-sulfatide is unknown. We show here that ASA-deficient murine macrophages and microglial cells express an endo-N-deacylase that cleaves the N-linked fatty acid from sulfatide. An ASA-deficient astrocytoma cell line devoid of this activity was used to identify the enzyme by overexpressing 13 deacylases with potentially matching substrate specificities. Hydrolysis of sulfatide was detected only in cells overexpressing the enzyme fatty acid amide hydrolase (FAAH). A cell-free assay with recombinant FAAH confirmed the novel role of this enzyme in sulfatide hydrolysis. Consistent with the in vitro data, deletion of FAAH lowered lyso-sulfatide levels in a mouse model of MLD. Regardless of the established cytotoxicity of lyso-sulfatide and the anti-inflammatory effects of FAAH inhibition seen in mouse models of several neurological diseases, genetic inactivation of FAAH did not mitigate, but rather exacerbated the disease phenotype of MLD mice. This unexpected finding was reflected by worsening of rotarod performance, increase of anxiety-related exploratory activity, aggravation of peripheral neuropathy, and reduced life expectancy. Thus, we conclude that FAAH has a protective function in MLD and may represent a novel therapeutic target for treatment of this fatal condition.

Substrate reduction therapy for Krabbe disease and metachromatic leukodystrophy using a novel ceramide galactosyltransferase inhibitor.

Krabbe disease (KD) and metachromatic leukodystrophy (MLD) are caused by accumulation of the glycolipids galactosylceramide (GalCer) and sulfatide and their toxic metabolites psychosine and lysosulfatide, respectively. We discovered a potent and selective small molecule inhibitor (S202) of ceramide galactosyltransferase (CGT), the key enzyme for GalCer biosynthesis, and characterized its use as substrate reduction therapy (SRT). Treating a KD mouse model with S202 dose-dependently reduced GalCer and psychosine in the central (CNS) and peripheral (PNS) nervous systems and significantly increased lifespan. Similarly, treating an MLD mouse model decreased sulfatides and lysosulfatide levels. Interestingly, lower doses of S202 partially inhibited CGT and selectively reduced synthesis of non-hydroxylated forms of GalCer and sulfatide, which appear to be the primary source of psychosine and lysosulfatide. Higher doses of S202 more completely inhibited CGT and reduced the levels of both non-hydroxylated and hydroxylated forms of GalCer and sulfatide. Despite the significant benefits observed in murine models of KD and MLD, chronic CGT inhibition negatively impacted both the CNS and PNS of wild-type mice. Therefore, further studies are necessary to elucidate the full therapeutic potential of CGT inhibition.

Impact of Long-Term Enzyme Replacement Therapy on Glucosylsphingosine (Lyso-Gb1) Values in Patients with Type 1 Gaucher Disease: Statistical Models for Comparing Three Enzymatic Formulations.

For three decades, enzyme replacement therapy (ERT), and more recently, substrate reduction therapy, have been the standard-of-care for type I Gaucher disease (GD1). Since 2012, three different ERTs have been available. No clinical trial or academic study has ever compared these ERTs beyond one year. Herein we compare the impact of the ERTs on repeated measurements of glucosylsphingosine (lyso-Gb1; the most sensitive and GD-specific biomarker). A total of 135 adult patients (77 (57%) female) with GD1, followed from July 2014 to March 2020 and treated with a single ERT (imiglucerase (n = 41, 30.4%), taliglucerase alfa (n = 21, 15.6%) and velaglucerase alfa (n = 73, 54.1%)), were included. Disease severity was defined by genotypes (mild: N370S (c.1226A>G) homozygous and N370S/R496H (c.1604G) compound heterozygous; severe: all other genotypes) and by the severity score index (SSI; mild: <7; severe: ≥7). Lyso-Gb1 testing was performed at Centogene™ on dry blood spot samples collected during routine visits. Patients treated with imiglucerase had higher lyso-Gb1 levels at different time points. A huge variation in lyso-Gb1 levels was noticeable both inter-individually and intra-individually for all three ERTs. A steeper and faster decrease of lyso-Gb1 levels was shown in velaglucerase alfa. Nevertheless, the differences between medications were not very large, and bigger numbers and more pretreatment data are required for more powerful conclusions.

Elevated glucosylsphingosine in Gaucher disease induced pluripotent stem cell neurons deregulates lysosomal compartment through mammalian target of rapamycin complex 1.

Gaucher disease (GD) is a lysosomal storage disorder caused by mutations in GBA1, the gene that encodes lysosomal ß-glucocerebrosidase (GCase). Mild mutations in GBA1 cause type 1 non-neuronopathic GD, whereas severe mutations cause types 2 and 3 neuronopathic GD (nGD). GCase deficiency results in the accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). GlcSph is formed by deacylation of GlcCer by the lysosomal enzyme acid ceramidase. Brains from patients with nGD have high levels of GlcSph, a lipid believed to play an important role in nGD, but the mechanisms involved remain unclear. To identify these mechanisms, we used human induced pluripotent stem cell-derived neurons from nGD patients. We found that elevated levels of GlcSph activate mammalian target of rapamycin (mTOR) complex 1 (mTORC1), interfering with lysosomal biogenesis and autophagy, which were restored by incubation of nGD neurons with mTOR inhibitors. We also found that inhibition of acid ceramidase prevented both, mTOR hyperactivity and lysosomal dysfunction, suggesting that these alterations were caused by GlcSph accumulation in the mutant neurons. To directly determine whether GlcSph can cause mTOR hyperactivation, we incubated wild-type neurons with exogenous GlcSph. Remarkably, GlcSph treatment recapitulated the mTOR hyperactivation and lysosomal abnormalities in mutant neurons, which were prevented by coincubation of GlcSph with mTOR inhibitors. We conclude that elevated GlcSph activates an mTORC1-dependent pathogenic mechanism that is responsible for the lysosomal abnormalities of nGD neurons. We also identify acid ceramidase as essential to the pathogenesis of nGD, providing a new therapeutic target for treating GBA1-associated neurodegeneration.

Detection of glucosylsphingosine in dried blood spots for diagnosis of Gaucher disease by LC-MS/MS.

INTRODUCTION: Gaucher disease (GD) is caused by a deficiency of ß-glucosidase (GCase), leading to accumulation of glucosylceramide (GlcC) and glucosylsphingosine (Lyso-Gb1). Lyso-Gb1 is a reliable biomarker for GD. OBJECTIVES: This study aims to develop a simple, effective and accurate method for the screening and diagnosis of GD using dried blood spot (DBS) samples. METHODS: Lyso-Gb1 in DBS was extracted by 50% acetonitrile aqueous solution containing isotope-labeled internal standard and analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). A reference interval was established by analyzing samples from 277 healthy controls. Lyso-Gb1 was detected in the residual DBS samples from 142 high-risk patients with splenomegaly and/or thrombocytopenia. Based on GCase activity in DBS, samples were classified into four groups: confirmed GD patients (n = 52), GD carriers (n = 5), false positive (n = 36) and negative (n = 49). RESULTS: The optimized Lyso-Gb1 assay showed intra- and inter-assay variations ranged between 2.0%-8.2% and 3.8%-10.2%, respectively. Accuracies ranged from 93.5% to 112.6%. The lowest limit of quantification was 1 ng/mL. The normal reference interval of Lyso-Gb1 in DBS ranged from 2.1 to 9.9 ng/mL. Among the 142 subjects, except for one GD patient (Lyso-Gb1 > 2500 ng/mL), the Lyso-Gb1 concentrations in 51 GD patients ranged from 190.5 to 2380.6 ng/mL (the median 614.8 ng/mL). Also, one negative patient was found to have an elevated Lyso-Gb1 level (684.5 ng/mL), while the other patients were normal. The negative case was then confirmed to be an atypical GD patient with a c.1091A > G (p.Y364C) homozygous variant in PSAP gene by next generation sequencing. CONCLUSIONS: The optimized method to determine Lyso-Gb1 in DBS was demonstrated as a useful tool for the screening and diagnosis of GD.

LC-MS/MS assays to quantify sulfatides and lysosulfatide in cerebrospinal fluid of metachromatic leukodystrophy patients.

Aim: Two separate LC-MS/MS assays were developed to quantitate sulfatides and lysosulfatide in human cerebrospinal fluid (CSF). Materials & methods: Lysosulfatide and the 15 most abundant sulfatide species were quantitated by LC-MS/MS using artificial CSF as surrogate matrix to prepare calibration curves. Results: Validation criteria were met (linear range: 0.02-1.00 µg/ml sulfatides [0.02-1.00 ng/ml lysosulfatide]); accuracy/precision were within ±15%. CSF from 21 children with metachromatic leukodystrophy had significantly higher sulfatide and lysosulfatide concentrations than CSF from 60 healthy children (p < 0.0001). Worse motor function correlated with higher CSF sulfatide (p = 0.0087) and lysosulfatide (p = 0.0034) levels. Conclusion: These assays, validated in patients with metachromatic leukodystrophy, may aid the clinical assessment of therapeutic responses.