Viable mouse models of acid beta-glucosidase deficiency: the defect in Gaucher disease

Abstract

Gaucher disease is an autosomal recessively inherited disease caused by mutations at the acid beta-glucosidase (GCase) locus (GBA). To develop viable models of Gaucher disease, point mutations (pmuts), encoding N370S, V394L, D409H, or D409V were introduced into the mouse GCase (gba) locus. DNA sequencing verified each unique pmut. Mutant GCase mRNAs were near wild-type (WT) levels. GCase activities were reduced to 2 to 25% of WT in liver, lung, spleen, and cultured fibroblasts from pmut/pmut or pmut/null mice. The corresponding brain GCase activities were approximately 25% of WT. N370S homozygosity was lethal in the neonatal period. For the other pmut mice, a few storage cells appeared in the spleen at > or =7 months (D409H or D409V homozygotes) or > or =1 year (V394L homozygotes). V394L/null, D409H/null, or D409V/null mice showed scattered storage cells in spleen at approximately 3 to 4 months. Occasional storage cells (sinusoidal cells) were present in liver. In D409V/null mice, large numbers of Mac-3-positive storage cells (ie, macrophages) accumulated in the lung. Glycosphingolipid analyses showed varying rates of progressive glucosylceramide accumulation in visceral organs of pmut/pmut or pmut/null mice, but not in brain. These GCase-deficient mice provide tools for gaining insight into the pathophysiology of Gaucher disease and developing improved therapies.

Figure 1.

Mouse gba targeting strategy. The WT gba is shown at the top. The replacement fragment from the targeting construct (second line) contained exons 5 to 11, parts of intron 4 and the 3′ flanking region, and a floxed neo selection marker in intron 8. The fourth line shows the resultant recombination with gba (gba with neo) and, following cre excision of neo (gba without neo) the recombined allele containing one loxP () site and the individual exon 9 mutations (*).

Figure 2.

Genotype analyses for gba point mutations. A 391 bp PCR fragment from WT (bottom arrow) or a 485-bp PCR fragment (391+loxP, top arrow) from point-mutated gba mice. The genotypes are indicated above each lane. +/+, WT homozygote; +/−, heterozygote (V394L, D409H, and D409V); and −/−, homozygote (eg, D409V). Ctrl is the PCR control without template DNA. MW = size standards.

Figure 3.

CBE inhibition of mouse N370S and V394L mGCases: WT (▾), V394L (♦) and N370S (▴) mGCase were harvested from media of Sf21 cells infected with the desired recombinant baculovirus. The enzymes were delipidated and dialyzed before being incubated (30 minutes; 37°C) with conduritol B epoxide (CBE, 1 to 500 μmol/L) and 4MU-Glc (4 mmol/L). The control sample (▪) was from media of uninfected Sf21 cells.

Figure 4.

Storage cells in spleen and liver of gba point-mutated mice. A: Representative samples from spleen of D409V/null mice. A large storage cell (arrow) is illustrated with prominent amounts of pale staining vacuolated cytoplasm. (H&E, magnification, ×400). B: A representative section from liver. A small cluster of pale staining storage cells are illustrated in the sinusoidal space (H&E, magnification, ×400).

Figure 5.

Storage cell staining with Mac-3. A: Section of wild-type lung with no accumulation of storage cells in the alveolar spaces. B: D409V/null lung with large storage cells (arrows) in the alveolar space. C: Wild-type lung stained for Mac-3. D: Mac-3 staining of lung from the D409V/null mouse indicating macrophage origin of the storage cells. Magnification: ×165 (A), ×250 (B), ×350 (C and D).

Figure 6.

Glycosphingolipid (GSL) profiles from various tissue of point-mutated gba mice. Cerebellum, liver, spleen, and lung (∼100 mg wet weight) were from gba mutant mice at various ages. The tissues were processed for thin-layer chromatography. The genotypes and ages are indicated only on the bottom for clarity of presentation. Migration standards for of sphingolipids are on the right: GluCer, glucosylceramide; GalCer, galactosylceramide; SO₄-GalCer, sulfogalactosylceramide; LacCer, lactosylceramide; THCer, ceramide trihexoside; Spm, sphingomyelin. The borate-impregnated plates were developed in chloroform/methanol/water (65/25/4) using borate-impregnated plates. Mature WT mice showed little variation in GSL levels in the respective tissues within the time frame of these experiments.