Model Of Gaucher Disease Research Articles

Bone marrow adipocytes alteration in an model of Gaucher disease

Bone marrow adipocytes alteration in an model of Gaucher disease

Characterization of the visceral and neuronal phenotype of 4L/PS-NA mice modeling Gaucher disease.

Gaucher disease is caused by a deficiency in glucocerebrosidase that can result in non-neuronal as well as neuronal symptoms. Common visceral symptoms are an increased organ size, specifically of the spleen, and glucosylceramide as well as glucosylsphingosine substrate accumulations as a direct result of the glucocerebrosidase deficiency. Neuronal symptoms include motor deficits and strong alterations in the cerebellum. To evaluate the effect of new compounds for the treatment of this devastating disease, animal models are needed that closely mimic the human phenotype. The 4L/PS-NA mouse as model of Gaucher disease is shown to present reduced glucocerebrosidase activity similar to human cases but an in-depth characterization of the model was still not performed. We therefore analyzed 4L/PS-NA mice for visceral alterations, motor deficits and also neuronal changes like glucocerebrosidase activity, substrate levels and neuroinflammation. A special focus was set at pathological changes of the cerebellum. Our results show that 4L/PS-NA mice have strongly enlarged visceral organs that are infiltrated by enlarged leukocytes and macrophages. Furthermore, animals present strong motor deficits that are accompanied by increased glucosylceramide and glucosylsphingosine levels in the brain, astrocytosis and activated microglia in the cortex and hippocampus as well as reduced calbindin levels in the cerebellum. The latter was directly related to a strong Purkinje cell loss. Our results thus provide a detailed characterization of the 4L/PS-NA mouse model over age showing the translational value of the model and validating its usefulness for preclinical efficiency studies to evaluate new compounds against Gaucher disease.

Mouse macrophage model of Gaucher disease for evaluating non-inhibitory chaperone candidates

Mouse macrophage model of Gaucher disease for evaluating non-inhibitory chaperone candidates

Alterations in the properties of the cell membrane due to glycosphingolipid accumulation in a model of Gaucher disease

Gaucher disease is a lysosomal storage disease characterized by the malfunction of glucocerebrosidase resulting in the accumulation of glucosylceramide and other sphingolipids in certain cells. Although the disease symptoms are usually attributed to the storage of undigested substrate in lysosomes, here we show that glycosphingolipids accumulating in the plasma membrane cause profound changes in the properties of the membrane. The fluidity of the sphingolipid-enriched membrane decreased accompanied by the enlargement of raft-like ordered membrane domains. The mobility of non-raft proteins and lipids was severely restricted, while raft-resident components were only mildly affected. The rate of endocytosis of transferrin receptor, a non-raft protein, was significantly retarded in Gaucher cells, while the endocytosis of the raft-associated GM1 ganglioside was unaffected. Interferon-γ-induced STAT1 phosphorylation was also significantly inhibited in Gaucher cells. Atomic force microscopy revealed that sphingolipid accumulation was associated with a more compliant membrane capable of producing an increased number of nanotubes. The results imply that glycosphingolipid accumulation in the plasma membrane has significant effects on membrane properties, which may be important in the pathogenesis of Gaucher disease.

New Macrophage Models of Gaucher Disease Offer New Tools for Drug Development

Gaucher disease is an inherited enzyme deficiency resulting in the lysosomal accumulation of specific glycolipids in macrophages and, in some cases, neurons. While current treatments are effective at reducing this glycolipid storage in macrophages, they are expensive and ineffective in treating neurological manifestations of the disease, driving the search for novel therapeutics. Moreover, mutations in GBA1, the gene implicated in Gaucher disease, are an important risk factor for the development of Parkinson disease and related disorders, an association that has further heightened interest in Gaucher disease research. However, the development of therapeutic strategies has been hampered by a shortage of appropriate cellular models of Gaucher disease. We have generated two novel macrophage models of Gaucher disease, one through the differentiation of peripheral blood monocytes from patients with Gaucher disease and the other through the differentiation of induced pluripotent stem cells derived from patient fibroblasts. Both disease models demonstrate similar cellular phenotypes and exhibit extensive glycolipid storage when exposed to exogenous lipid sources such as erythrocyte membranes. Furthermore, we have used these models to confirm the efficacy of a novel small molecule in clearing glycolipid storage and restoring normal macrophage function. These results demonstrate the usefulness of these models in exploring new therapeutics for Gaucher disease and related disorders.

C5a mediated increases of CD40 and CD40L molecules on dendritic and T cells are critical to Th1–Th17 mediated inflammation in Gaucher disease model

C5a mediated increases of CD40 and CD40L molecules on dendritic and T cells are critical to Th1–Th17 mediated inflammation in Gaucher disease model

The development and evaluation of a macrophage model of Gaucher disease

The development and evaluation of a macrophage model of Gaucher disease

Bone Marrow Microenvironment in an In Vitro Model of Gaucher Disease: Consequences of Glucocerebrosidase Deficiency

Gaucher disease (GD) is a lysosomal storage disorder due to glucocerebrosidase (GBA) deficiency. Mechanisms leading to the emergence of hematological and skeletal manifestations observed in GD are poorly explained. Bone marrow (BM) mesenchymal stem cells (MSCs) are multipotent progenitors that participate in the regulation of bone mass. MSCs should thus represent a cell population involved in the development or progression of bone disease in GD. In a chemical model of GD obtained with Conduritol β epoxide (CBE), a specific inhibitor of GBA activity, we functionally characterized BM MSCs and specifically analyzed their capacity to differentiate into osteoblasts. GBA deficiency obtained with CBE treatment, leads to a dramatic impairment of MSCs proliferation and to morphological abnormalities. Although the capacity of MSCs to differentiate into osteoblasts was not modified, the levels of several soluble factors that regulate bone metabolism were increased in MSCs treated with CBE, compared with untreated MSCs. Moreover, addition of conditioned media from CBE-treated MSCs on monocyte-derived osteoclasts cultured on bone matrix leads to an increase of resorption areas. These data suggested that, in GD, MSCs represents a stem cell population that is likely to be involved in bone pathogenesis.

Glucocerebrosidase deficiency dramatically impairs human bone marrow haematopoiesis in an in vitro model of Gaucher disease

One of the cardinal symptoms of type 1 Gaucher Disease (GD) is cytopenia, usually explained by bone marrow (BM) infiltration by Gaucher cells and hypersplenism. However, some cases of cytopenia in splenectomized or treated patients suggest possible other mechanisms. To evaluate intra-cellular glucocerebrosidase (GlcC) activity in immature progenitors and to prove the conduritol B epoxide (CBE)-induced inhibition of the enzyme, we used an adapted flow cytometric technique before assessing the direct effect of GlcC deficiency in functional assays. Among haematopoietic cells from healthy donors, monocytes showed the highest GlcC activity but immature CD34(+) and mesenchymal cells also had significant GlcC activity. CBE greatly inhibited the enzyme activity of all cell categories. GlcC-deficient CD34(+) cells showed impaired ability to proliferate and differentiate in the expansion assay and had lower frequency of erythroid burst-forming units, granulocyte colony-forming units (CFU) and macrophage CFU progenitors, but the effect of GlcC deficiency on megakaryocyte CFU lineage was not significant. GlcC deficiency strongly impaired primitive haematopoiesis in long-term culture. Furthermore, GlcC deficiency progressively impaired proliferation of mesenchymal progenitors. These data suggest an intrinsic effect of GlcC deficiency on BM immature cells that supplements the pathophysiology of GD and opens new perspectives of therapeutic approach.

11. Increasing expression of glucosylceramide-synthesizing enzyme in mouse models of Gaucher disease

11. Increasing expression of glucosylceramide-synthesizing enzyme in mouse models of Gaucher disease

45 Mouse models of Gaucher disease: Insights into visceral and CNS disease progression

45 Mouse models of Gaucher disease: Insights into visceral and CNS disease progression

Changes in macrophage morphology in a Gaucher disease model are dependent on CTP:phosphocholine cytidylyltransferase α

We have recently shown that phosphatidylcholine (PC) metabolism is altered in a macrophage model of Gaucher disease. We now demonstrate that treatment of macrophages with conduritol-B-epoxide (CBE), a glucocerebrosidase inhibitor, results in elevated activity of CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme in the pathway of PC biosynthesis. Furthermore, we provide evidence for a role for CCT in Gaucher macrophage growth by using macrophages derived from a genetically modified mouse which lacks a specific CCT isoform, CCTα, in macrophages. Upon CBE-treatment, macrophage size, analyzed by microscopy and by FACS, was significantly increased in macrophages from control mice, but did not increase, or increased to a much lower extent, in CCTα−/− macrophages. Together, these results suggest that the increase in PC biosynthesis is mediated via CCTα, and suggests a possible role for macrophage CCTα in Gaucher disease pathology.

Conditional expression of human acid β-glucosidase improves the visceral phenotype in a Gaucher disease mouse model

The reversibility and regression of histological and biochemical findings in a mouse model of Gaucher disease (4L/PS-NA) was evaluated using a liver-enriched activator protein promoter control of a tetracycline-controlled transcriptional activation-responsive human acid beta-glucosidase (hGCase) transgenic system. 4L/PS-NA has the acid beta-glucosidase (GCase) V394L/V394L (4L) point mutation combined with hypomorphic ( approximately 6% wild-type) expression of the mouse prosaposin transgene (PS-NA). The hGCase/4L/PS-NA had exclusive liver expression of hGCase controlled by doxycycline (DOX). In the absence of DOX, hGCase was secreted from liver at levels of approximately 120 microg/ml serum with only approximately 8% of full activity, following exposure to pH 7.4 in serum. The hGCase activity and protein were detected in cells of the liver (massive), lung, and spleen, but not the brain. The visceral tissue storage cells and glucosylceramide (GC) accumulation in hGCase/4L/PS-NA were decreased from that in 4L/PS-NA mice. Turning off hGCase expression with dietary DOX led to reaccumulation of storage cells and of GC in liver, lung, and spleen, and macrophage activation in those tissues. This study demonstrates that conditionally expressed hGCase supplemented the existing mutant mouse GCase to control visceral substrate accumulation in vivo.

1021. Gene Transfer Studies in a Novel Murine Model of Gaucher Disease

1021. Gene Transfer Studies in a Novel Murine Model of Gaucher Disease

37. Feasibility of In Vivo Gene Therapy in an Induced Murine Model of Gaucher Disease

37. Feasibility of In Vivo Gene Therapy in an Induced Murine Model of Gaucher Disease

N-butyldeoxygalactonojirimycin inhibits glycolipid biosynthesis but does not affect N-linked oligosaccharide processing.

We have previously reported that the imino sugar N-butyldeoxynojirimycin (NB-DNJ) inhibits glycolipid biosynthesis, in addition to its known activity as an inhibitor of the N-linked oligosaccharide processing enzyme alpha-glucosidase I. In an attempt to dissociate these two activities and identify an inhibitor which was more selective for the glycolipid biosynthetic pathway, several imino sugars have been N-alkylated and tested for inhibitory activity. The galactose analogue N-butyldeoxygalactonojirimycin (NB-DGJ) was found to be a potent inhibitor of glycolipid biosynthesis but in contrast to NB-DNJ had no effect on the maturation of N-linked oligosaccharides or on lysosomal glucocerebrosidase. The effect of increasing N-alkyl chain length on glycolipid inhibition was investigated. Nonalkylated DGJ, the N-methyl and N-ethyl derivatives, were noninhibitory. However, N-propylation resulted in partial inhibition while the N-butyl and N-hexyl derivatives resulted in maximal inhibition. Increasing alkyl chain length also resulted in increased potency of glucosyltransferase inhibition. In an in vitro Gaucher's disease model NB-DGJ was as effective as NB-DNJ in preventing glycolipid storage and may represent a more selective potential therapeutic agent than NB-DNJ for the management of this and other glycosphingolipidoses.

N-butyldeoxynojirimycin is a novel inhibitor of glycolipid biosynthesis.

The imino sugar deoxynojirimycin and its alkylated derivatives are inhibitors of the N-linked oligosaccharide processing enzymes alpha-glucosidase I and II. These compounds are glucose analogues and have the potential to inhibit both glucosidases and glucosyltransferases. However, to date there has been no report of deoxynojirimycin or similar analogues inhibiting a mammalian glucosyltransferase. We have investigated the effects of deoxynojirimycin and its alkylated derivatives on the biosynthesis of glycolipids in HL-60 cells. We have found that the N-butyl and N-hexyl derivatives of deoxynojirimycin, but not deoxynojirimycin itself, are novel inhibitors of the glucosyltransferase-catalyzed biosynthesis of glucosylceramide. This results in the inhibition of biosynthesis of all glucosylceramide-based glycosphingolipids. We have investigated the ability of one of these compounds, N-butyldeoxynojirimycin, to offset glucosylceramide accumulation in an in vitro Gaucher's disease model. This compound prevents lysosomal glycolipid storage and offers a novel therapeutic approach for the management of this and other glycolipid storage disorders.

Diagnosing sphingolipidoses in murine and human embryos

The aim of this study was to diagnose lipid storage diseases in embryos at the preimplantation stage. Two parallel approaches were employed. Firstly, activities of several sphingolipid hydrolases were determined in extracts of murine embryos and also human oocytes and polyspermic embryos. Sensitive fluorescent or fluorogenic procedures provided indications that Tay-Sachs, Gaucher and Krabbe diseases might be diagnosed in one human blastomere, while for Niemann-Pick disease two might be required. Secondly, pyrene lipids were administered into murine embryos and their fluorescence was quantified by computerized imaging microscopy. As a model of Gaucher disease, the fluorescent substrate pyrene glucosylceramide was administered into murine embryos in the presence or absence of an inhibitor of the enzyme beta-glucosidase. Because of decreased degradation of the substrate in enzyme-inhibited cells, the fluorescence per blastomere was considerably greater relative to those which received no inhibitor. The results indicated that lipid storage diseases might be diagnosed in single human blastomeres at the preimplantation stage, obviating the need for pre-natal diagnosis and abortion of affected foetuses.