Kate Zhang

Glucocerebrosidase gene-deficient mouse recapitulates Gaucher disease displaying cellular and molecular dysregulation beyond the macrophage

In nonneuronopathic type 1 Gaucher disease (GD1), mutations in the glucocerebrosidase gene (GBA1) gene result in glucocerebrosidase deficiency and the accumulation of its substrate, glucocerebroside (GL-1), in the lysosomes of mononuclear phagocytes. This prevailing macrophage-centric view, however, does not explain emerging aspects of the disease, including malignancy, autoimmune disease, Parkinson disease, and osteoporosis. We conditionally deleted the GBA1 gene in hematopoietic and mesenchymal cell lineages using an Mx1 promoter. Although this mouse fully recapitulated human GD1, cytokine measurements, microarray analysis, and cellular immunophenotyping together revealed widespread dysfunction not only of macrophages, but also of thymic T cells, dendritic cells, and osteoblasts. The severe osteoporosis was caused by a defect in osteoblastic bone formation arising from an inhibitory effect of the accumulated lipids LysoGL-1 and GL-1 on protein kinase C. This study provides direct evidence for the involvement in GD1 of multiple cell lineages, suggesting that cells other than macrophages may be worthwhile therapeutic targets.

The use of dried blood spot samples in the diagnosis of lysosomal storage disorders — Current status and perspectives

Dried blood spot (DBS) methods are currently available for identification of a range of lysosomal storage disorders (LSDs). These disorders are generally characterized by a deficiency of activity of a lysosomal enzyme and by a broad spectrum of phenotypes. Diagnosis of LSD patients is often delayed, which is of particular concern as therapeutic outcomes (e.g. enzyme replacement therapy) are generally more favorable in early disease stages. Experts in the field of LSDs diagnostics and screening programs convened and reviewed experiences with the use of DBS methods, and discuss the diagnostic challenges, possible applications and quality programs in this paper. Given the easy sampling and shipping and stability of samples, DBS has evident advantages over other laboratory methods and can be particularly helpful in the early identification of affected LSD patients through neonatal screening, high-risk population screening or family screening.

Glucocerebrosidase 2 gene deletion rescues type 1 Gaucher disease.

Significance Type 1 Gaucher disease (GD1) is a rare autosomal recessive disorder caused by inherited mutations in the glucocerebrosidase (GBA1) gene. This disease results in a marked accumulation of glycosphingolipid substrates, causing visceromegaly, cytopenia, and osteopenia. Here, we have rescued this clinical phenotype in GD1 mice by genetically deleting Gba2, a gene encoding a downstream extralysosomal enzyme, GBA2. We also report that sphingosine production in GD1 patients may contribute to the low-turnover bone loss. Our data suggest that GBA2 and sphingosine are potential targets for pharmacological intervention in GD1 patients. The inherited deficiency of the lysosomal glucocerebrosidase (GBA) due to mutations in the GBA gene results in Gaucher disease (GD). A vast majority of patients present with nonneuronopathic, type 1 GD (GD1). GBA deficiency causes the accumulation of two key sphingolipids, glucosylceramide (GL-1) and glucosylsphingosine (LysoGL-1), classically noted within the lysosomes of mononuclear phagocytes. How metabolites of GL-1 or LysoGL-1 produced by extralysosomal glucocerebrosidase GBA2 contribute to the GD1 pathophysiology is not known. We recently recapitulated hepatosplenomegaly, cytopenia, hypercytokinemia, and the bone-formation defect of human GD1 through conditional deletion of Gba in Mx1–Cre+:GD1 mice. Here we show that the deletion of Gba2 significantly rescues the GD1 clinical phenotype, despite enhanced elevations in GL-1 and LysoGL-1. Most notably, the reduced bone volume and bone formation rate are normalized. These results suggest that metabolism of GL-1 or LysoGL-1 into downstream bioactive lipids is a major contributor to the bone-formation defect. Direct testing revealed a strong inhibition of osteoblast viability by nanomolar concentrations of sphingosine, but not of ceramide. These findings are consistent with toxicity of high circulating sphingosine levels in GD1 patients, which decline upon enzyme-replacement therapy; serum ceramide levels remain unchanged. Together, complementary results from mice and humans affected with GD1 not only pinpoint sphingosine as being an osteoblast toxin, but also set forth Gba2 as a viable therapeutic target for the development of inhibitors to ameliorate certain disabling consequences of GD1.

Glucosylsphingosine is a key biomarker of Gaucher disease.

Gaucher disease (GD) involves the accumulation of glucosylceramide (GL1) and its deacylated lysolipid, glucosylsphingosine (lyso‐GL1) which is implicated in mediating immune dysregulation and skeletal disease. The aim of our study was to assess plasma Lyso‐GL1 as a biomarker of GD and its response to therapy.

An improved high-throughput dried blood spot screening method for Gaucher disease.

Gaucher disease is a lysosomal storage disorder caused by the deficiency of the lysosomal hydrolase glucocerebrosidase (β-glucocerebrosidase or acid β-D-glucosidase, GBA, EC 3.2.1.45). The deficiency of GBA leads to the accumulation of glucosylceramide (glucocerebroside) in the lysosomes of cells in the monocyte/ macrophage system. In Gaucher disease, glycosphingolipid-engorged cells displace normal cells in the liver, spleen and bonemarrow, which can lead to hepatosplenomegaly, thrombocytopenia, organ dysfunction, and skeletal deterioration [1]. The diagnosis of Gaucher disease is confirmed by measuring GBA activity in peripheral blood leukocytes; however, many patients are misdiagnosed or remain undiagnosed because Gaucher disease is rare and patients can have heterogeneous clinical symptoms [2]. The use of a simple screening method in high risk populations could increase the diagnostic rate of Gaucher disease and permit therapeutic intervention as needed to prevent serious complications. A high-throughput and reliable fluorescent assay was developed using the β-D-glucosidase substrate 4-MUG and CBE, an irreversible inhibitor of GBA that allows the distinction between GBA and other β-glucosidase isoenzymes [3]. Specimens from 20 previously diagnosed Gaucher disease patients were collected with written informed consent. Specimens from 193

Improved sensitivity of an acid sphingomyelinase activity assay using a C6:0 sphingomyelin substrate

Short-chain C6-sphingomyelin is an artificial substrate that was used in an acid sphingomyelinase activity assay for a pilot screening study of patients with Niemann–Pick disease types A and B. Using previously published multiplex and single assay conditions, normal acid sphingomyelinase activity levels (i.e. false negative results) were observed in two sisters with Niemann–Pick B who were compound heterozygotes for two missense mutations, p.C92W and p.P184L, in the SMPD1 gene. Increasing the sodium taurocholate detergent concentration in the assay buffer lowered the activity levels of these two patients into the range observed with other patients with clear separation from normal controls.

A Simple, High-Throughput Method for Analysis of Ceramide, Glucosylceramide, and Ceramide Trihexoside in Dried Blood Spots by LC/MS/MS

A unique monophasic extraction system coupled with LC/MS/MS to reduce matrix effects for sphingolipid analysis was developed. A solvent mixture of methanol, acetonitrile, and water was identified to simultaneously extract multiple sphingolipids with broad polarity range. To reduce matrix effects, the targeted sphingolipids were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The extraction solvent was used as an isocratic mobile phase in chromatographic separation to eliminate solvent exchange steps and enable high-throughput multiple lipid assay. The assay is linear for ceramide from 0.6 to 9 μg/mL with bias <15 %. The intra-assay coefficient of variation is less than 10 % for concentrations from 1.2 to 9 μg/mL, and less than 25 % for concentrations below 1.2 μg/mL. For glucosylceramide and ceramide trihexoside the linear range is 0.05-3 μg/mL with biases <10 % and <20 %, respectively. The intra-assay coefficient of variation for these analytes is less than 10 % at concentrations from 0.4 to 3 μg/mL, and less than 25 % for concentrations below 0.4 μg/mL.