The Developmental and Metabolic Neurology Branch was closed on January 31, 2009. This report reflects projects that were at or near completion by that date.? Basic Studies on Fabry Disease:? We have investigated the protein expression profiles in Fabry cells using microarray and proteomics technology. Gene expression analyses of the heart, aorta, and liver of male alpha-galactosidase A knockout mice before and after multiple injections of alpha-galactosidase A demonstrated an alteration of Rpgrip1. Enzyme replacement therapy tended to normalize gene expression. Future developments in array technology for proteins and DNA single nucleotide polymorphism analysis, together with gene expression microarray analysis, will open a new chapter in our understanding of the biology of lysosomal storage disorders. Globotriaosylceramide (Gb3) immunoreactivity in a heterogenous pattern was present in all organs examined of a patient on long-term enzyme replacement therapy. In the brain, immmunopositivity was found only in the parahippocampal region. Gb3 immunostaining was present in the cell membrane and cytoplasm of endothelial cells, even in the absence of lysosomal inclusions. In kidney tissue, Gb3 colocalized with lysosomal, endoplasmic reticulum, and nuclear markers. Pre- and postembedding immunogold electron microscopy of skin biopsies and untreated patient cultured skin fibroblasts confirmed the presence of Gb3 in the cell membrane, in various cytoplasmic structures, and in the nucleus. We concluded that a substantial amount of lysosomal and extralysosomal Gb3 immunoreactivity remains in cells and tissues even after years of enzyme replacement therapy in Fabry disease. These findings are crucial for the understanding of the disease mechanism and suggest the usefulness of immunostaining for Gb3 as a means to assess response to novel, specific therapies.? ? Cultured Endothelial Cell Model:? A cultured endothelial cell model with an extended lifespan was created as a critical element for future study of the vasculopathy of Fabry disease. The cell line has markedly extended lifespan compared to parental primary cell, continues to stably express many key markers of endothelial cells and retains many functional characteristics. The cells show significantly reduced activity of alpha-galactosidase A compared with primary endothelial cells from normal individuals and accumulate globotriaosylceramide in lysosomes. Gb3-loading resulted in increased intracellular reactive oxygen species (ROS) production in cultured vascular endothelial cells in a dose-dependent manner. Increased Gb3 also induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. Reduction of endogenous Gb3 by treatment of the cells with an inhibitor of glycosphingolipid synthase or alpha-galactosidase A led to decreased expression of adhesion molecules. Plasma from Fabry patients significantly increased ROS generation in endothelial cells when compared with plasma from non-Fabry controls. This effect was not influenced by reduction of intracellular Gb3. This study provided direct evidence that excess intracellular Gb3 induces oxidative stress and up-regulates the expression of cellular adhesion molecules in vascular endothelial cells. In addition, other factors in patient's plasma may also contribute to oxidative stress in Fabry vascular endothelial cells.? ? This cell line will continue to provide a useful in vitro model of Fabry disease and will facilitate systematic studies to investigate pathogenic mechanisms and explore new therapeutic approaches for Fabry disease.? ? ? Enzyme Replacement Therapy (ERT):? High affinity, slow-binding competitive inhibitors of glucocerebrosidase (GLA) were tested as potential adjunct therapy to improve effectiveness of enzyme replacement therapy (ERT). Preincubation of GLA with isofagomine (IFG), a slow-binding inhibitor, significantly increased stability of the enzyme to heat, neutral pH and denaturing agents in vitro. Preincubation of GLA with isofagomine prior to uptake by cultured cells results in increased intracellular enzyme activity accompanied by an increase in enzyme protein suggesting that reduced denaturation of GLA in the presence of isofagomine leads to a decrease in the degradation of the enzyme after internalization. We concluded that preincubation of GLA with slow-binding inhibitors before infusion may improve the effectiveness of ERT for Gaucher disease.? ? Pharmacological Chaperone Therapy: ? We developed a rapid screening assay for enhancement of endogenous alpha-galactosidase A (alpha-Gal A) in patient-derived cells. We used a T-cell based system to screen 40 mutations causing Fabry disease for enhanceability using 1-deoxygalactonojirimycin (DGJ). 17 mutations were found to be enhanceable to at least 25% of normal using this test system. Mutations located within alpha-helical or beta-sheets in the molecule were less likely to be enhanceable compared with mutations located in intervening sequences outside these regions. The level of activity achieved provides a basis for the therapeutic trial of DGJ in patients with similarly enhanceable enzyme. This assay method has general utility in other disorders in assessing the degree of enhancement of activity of mutated proteins by PCT and the identification of the pattern of enhanceability provides a rational basis for increased screening of patients who are more likely to benefit. Mapping the missense mutations to the structure of alpha-Gal A identified several factors that may influence response. Mutations in regions that are not in alpha-helix or beta-sheets, neither involved in disulfide bonds nor with an identified functional or structural role were more likely to respond. Predictability is, however, not precise and testing of each mutation for response to pharmacological chaperone therapy is necessary for Fabry disease and related lysosomal storage disorders.? ? Protein Transduction Domains: ? We have extended our strategies to broaden the organ and tissue distribution of exogenous enzymes for the treatment of both Gaucher disease and Fabry disease to augment the effectiveness of enzyme replacement therapy. We have previously expressed a TAT-GC fusion protein in which recombinant glucocerebrosidase (GC) is fused to 11-amino acid peptide from the HIV-1 transactivator protein (TAT) which functions to facilitate the transport of the enzyme across the plasma membrane of a variety of cell-types in a receptor-independent manner. We have extended these studies to include additional GC constructs containing flexible spacers to increase enzyme activity in addition to a variety of TAT analogs in order to test the effect of sequence variation on the uptake of the enzyme. Further studies have also been undertaken to construct similar fusions with the enzyme alpha-galactosidase A (AGA) as a possible strategy for wider distribution of this enzyme for the treatment of Fabry disease. Efforts are underway to introduce these fusion proteins into animals to test their distribution and compare these findings with that of the native constructs. Recombinant GC-TAT and AGA-TAT were expressed in eukaryotic cells from which catalytically active, normally glycosylated enzyme fusion proteins were obtained and tested for receptor independent uptake into cultured cells. It is expected that GC-TAT will be more efficiently delivered than unmodified GC to cells in the bone marrow and lung, and perhaps additional cells that lack the mannose lectin and thereby enhance the clinical responses of patients with Gaucher to enzyme replacement therapy. The AGA-TAT enzyme may be capable of entering heart and kidney more efficiently than the native enzyme. Alterations in the enzyme to improve the stability of the administered enzyme are currently under investigation.
| Shin, Sang H; Kluepfel-Stahl, Stefanie; Cooney, Adele M et al. (2008) Prediction of response of mutated alpha-galactosidase A to a pharmacological chaperone. Pharmacogenet Genomics 18:773-80 |
| Goldin, Ehud; Caruso, Rafael C; Benko, William et al. (2008) Isolated ocular disease is associated with decreased mucolipin-1 channel conductance. Invest Ophthalmol Vis Sci 49:3134-42 |
| Shen, Jin-Song; Meng, Xing-Li; Moore, David F et al. (2008) Globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in Fabry disease endothelial cells. Mol Genet Metab 95:163-8 |
| Shen, Jin-Song; Edwards, Nancy J; Hong, Young Bin et al. (2008) Isofagomine increases lysosomal delivery of exogenous glucocerebrosidase. Biochem Biophys Res Commun 369:1071-5 |
| Shin, Sang-Hoon; Murray, Gary J; Kluepfel-Stahl, Stefanie et al. (2007) Screening for pharmacological chaperones in Fabry disease. Biochem Biophys Res Commun 359:168-73 |
| Shen, Jin-Song; Meng, Xing-Li; Schiffmann, Raphael et al. (2007) Establishment and characterization of Fabry disease endothelial cells with an extended lifespan. Mol Genet Metab 92:137-44 |
| Askari, Hasan; Kaneski, Christine R; Semino-Mora, Cristina et al. (2007) Cellular and tissue localization of globotriaosylceramide in Fabry disease. Virchows Arch 451:823-34 |
| Moore, David F; Kaneski, Christine R; Askari, Hasan et al. (2007) The cerebral vasculopathy of Fabry disease. J Neurol Sci 257:258-63 |
| Lonser, R R; Schiffman, R; Robison, R A et al. (2007) Image-guided, direct convective delivery of glucocerebrosidase for neuronopathic Gaucher disease. Neurology 68:254-61 |
| Yoshimitsu, M; Higuchi, K; Ramsubir, S et al. (2007) Efficient correction of Fabry mice and patient cells mediated by lentiviral transduction of hematopoietic stem/progenitor cells. Gene Ther 14:256-65 |
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