Gaucher disease and Fabry disease: New markers and insights in pathophysiology for two distinct glycosphingolipidoses

Gaucher disease (GD) and Fabry disease (FD) are two relatively common inherited glycosphingolipidoses caused by deficiencies in the lysosomal glycosidases glucocerebrosidase and alpha-galactosidase A, respectively. For both diseases enzyme supplementation is presently used as therapy. Cells and tissues of GD and FD patients are uniformly deficient in enzyme activity, but the two diseases markedly differ in cell types showing lysosomal accumulation of the glycosphingolipid substrates glucosylceramide and globotriaosylceramide, respectively. The clinical manifestation of Gaucher disease and Fabry disease is consequently entirely different and the response to enzyme therapy is only impressive in the case of GD patients. This review compares both glycosphingolipid storage disorders with respect to similarities and differences. Presented is an update on insights regarding pathophysiological mechanisms as well as recently available biochemical markers and diagnostic tools for both disorders. Special attention is paid to sphingoid bases of the primary storage lipids in both diseases. The value of elevated glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease for diagnosis and monitoring of disease is discussed as well as the possible contribution of the sphingoid bases to (patho)physiology. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Long-term expression of the human glucocerebrosidase gene in vivo after transplantation of bone-marrow-derived cells transformed with a lentivirus vector

BACKGROUND: Gaucher disease is a lysosomal storage disorder resulting from a deficiency of glucocerebrosidase (GC). Recently, lentivirus vectors have been developed for efficient gene transfer into hematopoietic stem cells (HSCs). A recombinant lentivirus vector was used to evaluate the transduction of the human GC gene into murine bone-marrow-derived HSCs and its expression in their progeny. METHODS: Murine HSCs were transduced with lentivirus vector (lenti-EF-GC; MOI = 10-100). We transplanted female wild-type C57BL/6J mice with genetically modified male HSCs via the tail vein. RESULTS: We show that intravenous transplantation of transduced HSCs has therapeutic potential. Enzyme activity was increased two- to three-fold in various tissues, especially in the hematopoietic system. Numerous transplanted HSCs survived for 6 months and were shown by PCR to contain the provirus genes; the Y chromosome was identified by FISH analysis in the cells of female mouse recipients. CONCLUSIONS: The recombinant lentiviral vector transduces HSCs that are capable of long-term gene expression in vivo. This approach is potentially useful for the treatment of patents with Gaucher disease and other lysosomal storage disorders.     

Induction of osteoclastogenesis in an in vitro model of Gaucher disease is mediated by T cells via TNF-α

Gaucher disease is a lysosomal storage disorder caused by deficiency of glucocerebrosidase enzymatic activity leading to accumulation of its substrate glucocerebrosidase mainly in macrophages. Skeletal disorder of Gaucher disease is the major cause of morbidity and is highly refractory to enzyme replacement therapy. However, pathological mechanisms of bone alterations in Gaucher disease are still poorly understood. We hypothesized that cellular alteration in Gaucher disease produces a proinflammatory milieu leading to bone destruction through enhancement of monocyte differentiation to osteoclasts and osteoclasts resorption activity. Against this background we decided to investigate in an in vitro chemical model of Gaucher disease, the capacity of secreted soluble mediators to induce osteoclastogenesis, and the mechanism responsible for this phenomena. We demonstrated that soluble factors produced by CBE-treated PBMC induced differentiation of osteoclasts precursors into mature and active osteoclasts that express chitotriosidase and secrete proinflammatory cytokines. We also showed a role of TNF-α in promoting osteoclastogenesis in Gaucher disease chemical model. To analyze the biological relevance of T cells in osteoclastogenesis of Gaucher disease, we investigated this process in T cell-depleted PBMC cultures. The findings suggest that T cells play a role in osteoclast formation in Gaucher disease. In conclusion, our data suggests that in vitro GCASE deficiency, along with concomitant glucosylceramide accumulation, generates a state of osteoclastogenesis mediated in part by pro-resorptive cytokines, especially TNF-α. Moreover, T cells are involved in osteoclastogenesis in Gaucher disease chemical model.     

4-Phenylbutyrate rescues trafficking incompetent mutant alpha-galactosidase A without restoring its functionality

Fabry disease is a lysosomal storage disorder caused by deficiency of alpha-galactosidase A. Most mutant enzyme is catalytically active but due to misfolding retained in the endoplasmic reticulum. We have tested 4-phenylbutyrate for its potential to rescue various trafficking incompetent mutant alpha-galactosidase A. Although we found that the trafficking blockade for endoplasmic reticulum-retained mutant alpha-Gal A was released, neither a mature enzyme was detectable in transgenic mice fibroblasts nor a reversal of lysosomal Gb3 storage in fibroblasts from Fabry patients could be observed. Because of lack of functionality of rescued mutant alpha-galactosidase A, 4-phenylbutyrate seems to be of limited use as a chemical chaperone for Fabry disease.     

Expression and secretion of human glucocerebrosidase mediated by recombinant lentivirus vectors in vitro and in vivo: implications for gene therapy of Gaucher disease

Gaucher disease is a lysosomal storage disorder resulting from a deficiency of glucocerebrosidase (GC). In this study, we showed that vascular and hepatic delivery of a HIV-1-based lentivirus vector encoding human GC cDNA produced therapeutic levels of GC protein. A high level of expression of GC was produced in cultured fibroblasts derived from patients with Gaucher disease by transducing the cells with recombinant lentivirus vectors. GC secreted by transduced fibroblasts was taken up by adjacent GC-deficient cells by endocytosis. Intraportal administration of lenti-EF-GC viral vector resulted in efficient transduction and expression of the GC. Vascular delivery of vector resulted in high levels of GC expression in mice that persisted in most organs over the four months. No significant abnormalities were found attributable to recombinant lentivirus vectors in any of the tissues examined. This study represents an initial step toward gene transfer using recombinant lentivirus vectors for treatment of Gaucher disease.     

Progress and potential of non-inhibitory small molecule chaperones for the treatment of Gaucher disease and its implications for Parkinson disease

Gaucher disease, caused by pathological mutations GBA1, encodes the lysosome-resident enzyme glucocerebrosidase, which cleaves glucosylceramide into glucose and ceramide. In Gaucher disease, glucocerebrosidase deficiency leads to lysosomal accumulation of substrate, primarily in cells of the reticulo-endothelial system. Gaucher disease has broad clinical heterogeneity, and mutations in GBA1 are a risk factor for the development of different synucleinopathies. Insights into the cell biology and biochemistry of glucocerebrosidase have led to new therapeutic approaches for Gaucher disease including small chemical chaperones. Such chaperones facilitate proper enzyme folding and translocation to lysosomes, thereby preventing premature breakdown of the enzyme in the proteasome. This review discusses recent progress in developing chemical chaperones as a therapy for Gaucher disease, with implications for the treatment of synucleinopathies. It focuses on the development of non-inhibitory glucocerebrosidase chaperones and their therapeutic advantages over inhibitory chaperones, as well as the challenges involved in identifying and validating chemical chaperones.     

Knockdown of chimeric glucocerebrosidase by green fluorescent protein-directed small interfering RNA

Gaucher disease, the most common type of lysosomal storage disorder, is characterized by an inherited deficiency of the membrane-associated hydrolase, glucocerebrosidase. Glucocerebrosidase catalyzes the hydrolysis of glucocerebroside to ceramide and glucose, a crucial step in the recycling of membrane sphingolipids. The exorbitant cost of the current treatment standard for Gaucher disease, enzyme replacement therapy, prevents many from receiving treatment. This limitation has led to a wide-spread search for more efficient and cost-effective methods of protein production and alternate therapies, resulting in a closer examination of glucocerebrosidase biosynthesis and current treatment techniques. The use of specific small interfering RNAs (siRNAs) to knock down target genes is an attractive option for studying such processes, though a glucocerebrosidase-specific siRNA has yet to be reported. We note, however, that green fluorescent protein (GFP)-directed siRNAs can not only provide a positive control to test siRNA delivery and system integrity, but also serve as a means to knock down a fusion partner without having to design siRNAs specific to the partner. After effectively co-transfecting COS-1 cells with enhanced GFP (EGFP)-tagged glucocerebrosidase constructs and GFP-directed siRNAs, we report successful knockdown of all EGFP-containing constructs at both the RNA and protein levels. This provides a method of examining enzyme biosynthesis and treatment options. Furthermore, this technique is applicable to other systems, since we have demonstrated the usefulness of GFP as a siRNA target in mammalian cells when fused to another gene of interest.     

A chimeric mouse model of Gaucher disease

BACKGROUND: There is a major need for a mouse model of Gaucher disease, but the glucocerebrosidase knockout mouse is not viable; it dies shortly before or immediately after birth, apparently because of involvement of the central nervous system and/or skin. The most common form of Gaucher disease, type I, has a phenotype that is limited to the monocyte-macrophage system. MATERIALS AND METHODS: We have created a chimeric mouse by infusing hematopoietic stem cells from fetuses that are homozygous for the glucocerebrosidase knockout into irradiated mice. RESULTS: The chimeric mice manifested a severe deficiency of glucocerebrosidase activity in peripheral blood cells and spleen indicating a lack of cell-cell correction. Levels of glucocerebroside in spleen and liver are increased, and infusing the mice with exogenous glucocerebroside/albumin particles produced a marked increase in the amount of glucocerebroside stored in liver and spleen. Morphologically identifiable Gaucher cells were not present. CONCLUSIONS: The chimeric model reflects the increased glycolipid storage in the reticuloendothelial system that is characteristic of Gaucher disease, and could be useful as a model for studying treatment of Gaucher disease.     

Gaucher's and Fabry's diseases: biochemical and genetic aspects

Gaucher and Fabry's diseases are lysosomal storage disorders. They are due to glucocerebrosidase or alpha galactosidase deficiency, respectively. Gaucher disease, transmitted as an autosomal recessive trait, is frequent among Ashkenazi Jews. Cloning of the gene has allowed the characterization of few common mutations. Some of them have a prognosis value, in favour of either a non neurological form (type 1) or more severe forms (types 2 and 3). There mutations were found in 70% of the alleles, the other alleles carrying private mutations. Fabry disease is transmitted as an X-linked recessive trait. Genetic counselling in at-risk families relies on the detection of carrier females. As the alpha galactosidase gene shows various mutations, the establishment of phenotype-genotype correlations is limited. These two diseases, well defined at the biochemical and genetic level, are good models of inherited diseases for the development of specific therapies.     

Failure of alglucerase infused into Gaucher disease patients to localize in marrow macrophages.

BACKGROUND: Gaucher disease is a common glycolipid storage disease, caused by a deficiency of lysosomal beta-glucosidase (glucocerebrosidase). Alglucerase is a form of glucocerebrosidase enriched with terminal mannose moieties, so as to "target" the preparation to the high-affinity macrophage receptor in patients with Gaucher disease. Our earlier in vitro studies indicated that alglucerase was bound by cells other than macrophages by a widely distributed, low-affinity mannose receptor. MATERIALS AND METHODS: Bone was removed at surgery from six patients with Gaucher disease; in three cases, bone was obtainable both when the patient was untreated and after receiving an infusion of alglucerase. Four samples of bone were obtained from patients without Gaucher disease and served as controls. A bone marrow aspirate was obtained from another patient with Gaucher disease immediately after enzyme infusion. Marrow beta-glucosidase activity and chitotriosidase (a macrophage marker) was determined on all samples. RESULTS: Even with the large bolus doses used for the treatment of Gaucher disease by some, scarcely any beta-glucosidase activity was found in marrow samples; the amount of the enzyme was much less than would have been anticipated had the enzyme been evenly distributed to all body cells. CONCLUSIONS: Alglucerase is not targeted to marrow macrophages. Its unquestioned therapeutic effectiveness must be due either to its activity at some site other than marrow macrophages or to the fact that the doses administered are so enormous that even a small fraction is sufficient to achieve a therapeutic effect.     

Comparative study on glucocerebrosidase in spleens from patients with Gaucher disease.

In Gaucher disease (glucosylceramide lipidosis), deficiency of glucocerebrosidase causes pathological storage of glucosylceramide, particularly in the spleen. A comparative biochemical and immunological analysis has therefore been made of glucocerebrosidase in spleens from normal subjects (n = 4) and from Gaucher disease patients with non-neuronopathic (n = 5) and neuronopathic (n = 5) phenotypes. The spleens from all Gaucher disease patients showed markedly decreased glucocerebrosidase activity. Discrimination of different phenotypes of Gaucher disease was not possible on the basis of the level of residual enzyme activity, or by measurements, using the immunopurified enzyme, of kinetic constants, pI or molecular mass forms. A severe decrease was found in the specific activity of glucocerebrosidase purified to homogeneity from the spleen of a patient with the non-neuronopathic phenotype of Gaucher disease, as compared with that of the enzyme purified from the spleen of a normal subject. This finding was confirmed by an immunological method developed for accurate assessment of the relative enzyme activity per molecule of glucocerebrosidase protein. The method revealed that the residual enzyme in the spleens of all investigated patients with a non-neuronopathic course of Gaucher disease had a more than 7-fold decreased activity of glucocerebrosidase (measured in the presence of taurocholate) per molecule of enzyme, and that the concentration of glucocerebrosidase molecules in the spleens of these patients was near normal. Observations made with immunoblotting experiments were consistent with these findings. In contrast, in the spleens of patients with neuronopathic phenotypes of Gaucher disease, the concentration of glucocerebrosidase molecules was severely decreased.     

Biochemistry of glycosphingolipid storage disorders: implications for therapeutic intervention

The physiological importance of the degradative processes in lysosomes is revealed by the existence of at least 40 distinct inherited diseases, the so-called lysosomal storage disorders. Most of these diseases are caused by a deficiency in a single lysosomal enzyme, or essential cofactor, and result in the lysosomal accumulation of one, or sometimes several, natural compounds. The most prevalent subgroup of the lysosomal storage disorders is formed by the sphingolipidoses, inherited disorders that are characterized by excessive accumulation of one or multiple (glyco)sphingolipids. The biology of glycosphingolipids has been extensively discussed in other contributions during this symposium. This review will therefore focus in depth on (type 1) Gaucher disease, a prototypical glycosphingolipidosis. The elucidation of the primary genetic defect, being a deficiency in the lysosomal glucocerebrosidase, is described. Characterization of glucocerebrosidase at protein and gene level has subsequently opened avenues for therapeutic intervention. The development of successful enzyme replacement therapy for type 1 Gaucher disease is discussed. Attention is also paid to the alternative approach of substrate modulation using orally administered inhibitors of glucosylceramide synthesis. Novel developments about the monitoring of age of onset, progression and correction of disease are described. The remaining challenges about pathophysiology of glycosphingolipidoses are discussed in view of further improvements in therapy for these debilitating disorders.     

Enzyme replacement therapy: conception,chaos and culmination

Soon after the enzymatic defects in Gaucher disease and in Niemann-Pick disease were discovered, enzyme replacement or enzyme supplementation was proposed as specific treatment for patients with these and related metabolic storage disorders. While relatively straightforward in concept, successful implementation of this approach required many years of intensive effort to bring it to fruition. Procedures were eventually developed to produce sufficient quantities of the requisite enzymes for clinical trials and to target therapeutic enzymes to lipid-storing cells. These achievements led to the development of effective enzyme replacement therapy for patients with Gaucher disease and for Fabry disease. These demonstrations provide strong incentive for the application of this strategy for the treatment of many human disorders of metabolism.     

Expression and characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A produced in Pichia pastoris

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A. This enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids. An improved method of production of recombinant alpha-galactosidase A for use in humans is needed in order to develop new approaches for enzyme therapy. Human alpha-galactosidase A for use in enzyme therapy has previously been obtained from human sources and from recombinant clones derived from human cells, CHO cells, and insect cells. In this report we describe the construction of clones of the methylotrophic yeast Pichia pastoris that produce recombinant human alpha-galactosidase A. Recombinant human alpha-galactosidase A is secreted by these Pichia clones and the level of production is more than 30-fold greater than that of previously used methods. Production was optimized using variations in temperature, pH, cDNA copy number, and other variables using shake flasks and a bioreactor. Expression of the human enzyme increased with increasing cDNA copy number at 25 degrees C, but not at the standard growth temperature of 30 degrees C. The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (POROS 20 CM, POROS 20 HQ) and hydrophobic (Toso-ether, Toso-butyl) chromatography with a BioCAD HPLC Workstation. Purified recombinant alpha-galactosidase A was taken up by fibroblasts derived from Fabry disease patients and normal enzyme levels could be restored under these conditions. Analysis of the carbohydrate present on the recombinant enzyme indicated the predominant presence of N-linked high-mannose structures rather than complex carbohydrates.     

Purification and characterization of human alpha-galactosidase A expressed in insect cells using a baculovirus vector

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A. The enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids. To perform more extensive biochemical characterization and to develop new approaches for enzyme therapy, a method of producing and purifying recombinant alpha-galactosidase A suitable for scale-up manufacture for use in humans is needed. Previously, a catalytically active recombinant human alpha-galactosidase A was expressed using a baculovirus vector and purified using conventional chromatography. However, the level of expression was too low to permit economical production and the chromatographic techniques used for enzyme purification were not suitable for enzyme to be used in humans. Therefore, the cDNA of the enzyme was cloned to an improved baculovirus vector and the enzyme was expressed in a 15-liter bioreactor using optimized growth conditions. Infection of insect cells by the baculovirus resulted in a significant fivefold increase in the level of secreted recombinant alpha-galactosidase A activity that is compatible with economic manufacturing. The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (Poros 20-CM, Poros 20-HQ) and hydrophobic chromatography (Toso-ether, Toso-butyl) using the BioCAD HPLC workstation. These chromatographic steps are readily scalable to larger volumes and are appropriate for the purification of the recombinant human alpha-galactosidase A to be used in clinical trials of enzyme replacement therapy for Fabry disease patients.     

Fabry disease--a metabolic disorder with a challenge for endocrinologists?

OBJECTIVE: To revisit Fabry disease, a rare X-linked metabolic glycosphingolipid storage disease caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). METHOD: Summary of the existing knowledge of Fabry disease including the clinical feature of Fabry disease and the recent breakthrough in the treatment of Fabry patients with the development of recombinant human alpha-gal A. CONCLUSION: The diffuse organ manifestations of Fabry disease resemble medical endocrinological diseases, and medical endocrinology might be an appropriate speciality to manage the treatment in collaboration with other specialists and clinical geneticists.     

AAV8-mediated expression of glucocerebrosidase ameliorates the storage pathology in the visceral organs of a mouse model of Gaucher disease

BACKGROUND: Gaucher disease is the most common of the lysosomal storage disorders. The primary manifestation is the accumulation of glucosylceramide (GL-1) in the macrophages of liver and spleen (Gaucher cells), due to a deficiency in the lysosomal hydrolase glucocerebrosidase (GC). A Gaucher mouse model (D409V/null) exhibiting reduced GC activity and accumulation of GL-1 was used to evaluate adeno-associated viral (AAV)-mediated gene therapy. METHODS: A recombinant AAV8 serotype vector bearing human GC (hGC) was administered intravenously to the mice. The levels of hGC in blood and tissues were determined, as were the effects of gene transfer on the levels of GL-1. Histopathological evaluation was performed on liver, spleen and lungs. RESULTS: Vector administration to pre-symptomatic Gaucher mice resulted in sustained hepatic secretion of hGC at levels that prevented GL-1 accumulation and the appearance of Gaucher cells in the liver, spleen and lungs. AAV administration to older mice with established disease resulted in normalization of GL-1 levels in the spleen and liver and partially reduced that in the lung. Analysis of the bronchoalveolar lavage fluid (BALF) from treated mice showed significant correction of the abnormal cellularity and cell differentials. No antibodies to the expressed hGC were detected following a challenge with recombinant enzyme suggesting the animals were tolerized to human enzyme. CONCLUSIONS: These data demonstrate the effectiveness of AAV-mediated gene therapy at preventing and correcting the biochemical and pathological abnormalities in a Gaucher mouse model, and thus support the continued consideration of this vector as an alternative approach to treating Gaucher disease.     

Uptake of Mannose-Terminal Glucocerebrosidase in Cultured Human Cholinergic and Dopaminergic Neuron Cell Lines

Enzyme replacement therapy has been shown to be particularly effective for patients with type 1 (non-neuronopathic) Gaucher disease. However, intravenously administered glucocerebrosidase does not reverse or halt the progression of brain damage in patients with type 2 (acute neuronopathic) Gaucher disease. A previous investigation revealed that intracerebral infusion of mannose-terminal glucocerebrosidase was safe in experimental animals. The enzyme had a comparatively long half-life in the brain. It was transported by convection from the site of infusion along white matter fiber tracts to the cerebral cortex where it was endocytosed by neurons. In anticipation of intracerebral administration of mannose-terminal glucocerebrosidase to patients with type 2 Gaucher disease, it was important to learn the mechanism involved in its cellular uptake. We therefore compared the endocytosis of this enzyme by J774 macrophage cells with that in two human neuronal cell lines and a human astrocyte cell line. Mannose-terminal glucocerebrosidase was taken up by cholinergic LA-N-2 cells, but to a much lower extent than by macrophages. Considerably less of the enzyme was endocytosed by dopaminergic SH-SY5Y cells. It was not taken up by NHA astrocytes. The findings provide encouragement for an exploration of intracerebral administration of glucocerebrosidase in patients with type 2 Gaucher disease.     

Delivery,Distribution, and Neuronal Uptake of Exogenous Mannose-Terminal Glucocerebrosidase in the Intact Rat Brain

Gaucher disease is caused by insufficient activity of the enzyme glucocerebrosidase. Great benefit has been obtained through enzyme replacement therapy for patients with type 1 (non-neuronopathic) Gaucher disease. In contrast, inconsistent effects of enzyme therapy have been observed in patients with type 3 (chronic neuronopathic) Gaucher disease, and no benefit on the lethal course of the disease occurs in patients with Type 2 (acute neuronopathic) Gaucher disease. We examined the use of convection-enhanced delivery to augment the delivery and distribution of exogenous glucocerebrosidase (m.w. 63,000) to the brain by infusing it under slight hydrostatic pressure into the striatal region of rats. The enzyme was comparatively stable under these conditions. It was distributed from the site of injection toward the cerebral cortex where it became primarily localized in neurons. These findings provide considerable incentive for the exploration of intracerebral microinfusion of enzyme to the brain of patients with metabolic storage disorders involving the CNS.