alpha-Galactosidase A deficient mice: a model of Fabry disease

Abstract

Fabry disease is an X-linked inherited metabolic disorder that is caused by a deficiency of alpha-galactosidase A (alpha-Gal A). Progressive deposition of neutral glycosphingolipids that have terminal a-linked galactosyl moieties in vascular endothelial cells causes renal failure along with premature myocardial infarctions and strokes in patients with this condition. No specific treatment is available for patients with this disorder at this time. An animal model of this condition would be valuable for exploring therapeutic strategies for patients with Fabry disease. We report here the generation of alpha-Gal A deficient mice by gene targeting and an analysis of the resulting phenotype. The knockout mice display a complete lack of alpha-Gal A activity. The mice, however, appeared clinically normal at 10 weeks of age. Ultrastructural analysis revealed concentric lamellar inclusions in the kidneys, and confocal microscopy using a fluorescent-labeled lectin specific for alpha-D-galactosyl residues showed accumulation of substrate in the kidneys as well as in cultured fibroblasts. Lipid analysis revealed a marked accumulation of ceramidetrihexoside in the liver and the kidneys. These findings indicate the similarity of the pathophysiological process in the mutant mice and in patients with Fabry disease. The deficiency of alpha-Gal A activity and the accumulation of material containing terminal alpha-galactosyl residues in cultured embryonic fibroblasts derived from alpha-Gal A(-/0) mice were corrected by transducing these cells with bicistronic multidrug resistance retroviruses containing human alpha-Gal A cDNA.

Figure 1

Targeted disruption of the α-Gal A gene in mouse ES cells and generation of α-Gal A(−/0) mice. (A) Schematic representation of the targeting construct (TC) and the wild-type (WT) and mutated (M) alleles of α-Gal A gene. The hatched bars indicate 5′ flanking probe (a) and neomycin resistance gene (neo) probe (b) used to identify targeted clones. Restriction enzyme sites are as follows: K, KpnI; B, BamHI; E, EcoRI; S, SacI. (B) A representative Southern blot analysis of tail DNA from α-Gal A(+/0), α-Gal A(+/−), and α-Gal A(−/0) mice hybridized with probe a. (C) α-Gal A activity in liver homogenates from α-Gal A(+/0) (n = 6) and α-Gal A(−/0) (n = 5) mice. Activities are expressed as nmol/h per mg protein. Bar = SD.

Figure 2

Electron micrographs of kidneys from a 10-week-old α-Gal A(−/0) mouse. (A) Inclusions are seen in lysosomes of the renal tubular cells (arrows) (×6250). (B) Lysosomes contain inclusions of concentric lamellar structures (×20,000). (C) Higher magnification of inclusions in B (×60,000).

Figure 3

FITC-labeled Bandeiraea simplicifolia lectin staining of kidneys from α-Gal A(+/0) (A and B) and α-Gal A(−/0) mice (C and D). (A) Kidney sections from a α-Gal A(+/0) mouse stained with FITC-labeled lectin. (B) Section as in A stained in the presence of 200 mM galactose. (C) Kidney section from a α-Gal A (−/0) mouse stained with FITC-labeled lectin. (D) Section as in C stained in the presence of 200 mM galactose. (Bar = 10 μm.)

Figure 4

FITC-labeled lectin staining of embryonic fibroblasts from α-Gal A(+/0) (A and B) and α-Gal A(−/0) (C–F) mice. Slides were incubated with lectin only (A, C, and E) or lectin and galactose (B, D, and F). (D) After transducing with MDR-based retrovirus (Ha-MDR-IRES-αGal), the KMG-2 clone showed the correction of metabolic deficit as seen by clearance of accumulated material (E). (Bars = 10 μm.)

Figure 5

Accumulation of CTH in liver and kidney from α-Gal A(−/0) mice. Liver and kidneys from wild-type (+/0, n = 2) and α-Gal A-deficient (−/0, n = 2) 10-week-old mice were analyzed by HPTLC as described. Purified porcine CTH (5 μg; Matreya, Pleasant Gap, PA) was used as a marker.