Combination of acid β-glucosidase mutation and Saposin C deficiency in mice reveals Gba1 mutation dependent and tissue-specific disease phenotype

Abstract

Gaucher disease is caused by mutations in GBA1 encoding acid β-glucosidase (GCase). Saposin C enhances GCase activity and protects GCase from intracellular proteolysis. Structure simulations indicated that the mutant GCases, N370S (0 S), V394L (4L) and D409V(9V)/H(9H), had altered function. To investigate the in vivo function of Gba1 mutants, mouse models were generated by backcrossing the above homozygous mutant GCase mice into Saposin C deficient (C*) mice. Without saposin C, the mutant GCase activities in the resultant mouse tissues were reduced by ~50% compared with those in the presence of Saposin C. In contrast to 9H and 4L mice that have normal histology and life span, the 9H;C* and 4L;C* mice had shorter life spans. 9H;C* mice developed significant visceral glucosylceramide (GC) and glucosylsphingosine (GS) accumulation (GC»GS) and storage macrophages, but lesser GC in the brain, compared to 4L;C* mice that presents with a severe neuronopathic phenotype and accumulated GC and GS primarily in the brain. Unlike 9V mice that developed normally for over a year, 9V;C* pups had a lethal skin defect as did 0S;C* mice resembled that of 0S mice. These variant Gaucher disease mouse models presented a mutation specific phenotype and underscored the in vivo role of Saposin C in the modulation of Gaucher disease.

Figure 1

Modeling of mutation effect on GCase structure. (A) GCase structure showing side chain interaction with D409. (B) Enlarged side chain interaction region. Red clouds show electronic force field at position 409. WT D409 at pH7.2 or pH 5.5 maintain the same side chain interactions with 7 surrounding amino acids showing green/yellow at pH 7.2 and white/blue at pH5.5. Carbons on amino acid are labeled as red dots. D409H gains additional side chain interactions at pH7.2 and turns more dramatic alterations in its conformation at pH 5.5. D409V interact with surrounding amino acids. This mutation at 409 changes D to V (Valine), a non-polar amino acid side chain, which lost all WT interactions with surrounding amino acid side chains. Human PDB crystal structures 2F61, pH7.2, 2.5 Å and 3GXI, pH 5.5, 1.84 Å from Swiss PDB Viewer (DeepView, SPDBV,Version 4.10) program were used for modelling. Amino acids involved side chain interaction are listed in Supplementary Table 1.

Figure 2

CNS pathology in 9H;C* mice. (A) Phenotype. 9H;C* mice showed hind-limb clasping during tail hanging at 3 months of age (left panel) and kyphotic posturing at 11 months of age (Right panel). As a control, C+/− mouse did not show hind-limb clasping. (B) CNS pathology in 9H;C* mice compared to WT;C* and 9H;C+/− control mice at 12 months of age. (Top panels) Loss of Purkinje cells (H&E, arrows) was evident in 9H;C* and WT;C* cerebellum and was accompanied with activated microglial cells positive for anti CD68 antibody (CD68, brown) staining. (Middle panels) Dorsal root ganglion in 9H;C* mice contained foamy storage materials in cells (H&E, arrows) and had CD68 positive cells (brown). (Lower panels) Dorsal horn of spinal cord in 9H;C* mice had axonal spheroids (H&E, arrows) and CD68 positive cells (brown). WT;C* mice had fewer foamy cells, axonal spheroids and CD68 positive cells than 9H;C* mice. As a control, 9H;C+/− mice tissues showed normal histology. (C) CD68 positive signals (brown) distributed differently in 9H;C* and 4L;C* brains. CD68 signals were restricted in caudate putamen (cp), thalamus (th) and cerebellum (cb) regions (arrows) in 9H;C* brain and distributed in most regions in 4L;C* brain.

Figure 3

Visceral pathology of 9H;C* mice. (A) H&E stained 9H;C* liver and lung at 13 months of age showed storage cells (arrows) in the liver (A) and lung (C). Ultrastructural studies demonstrated the storage cells form multi nucleic cluster in the liver (B). The storage materials had tubule form (B insert). The membrane inclusions were in the lung storage cells (D). (B) Anti-CD68 antibody (brown) stained liver and lung. 12-month WT mouse liver (A) and lung (B) showed background level of CD68 signals. 9H;C* liver (C) and lung (D) at 12 months of age had engorged CD68 positive macrophages (arrows). 4L;C* liver (E) and lung (F) at 45 days of age did not have storage cells. Scale bar = 100 µm for all images. (C) Compared to age-matched WT spleen stained by H&E (A) and anti-CD68 (B), 9H;C* spleen at 12 months of age had storage cells (arrows) by H&E (C) and CD68 positive cells (D).

Figure 4

Skin of 9V;C* and 0S;C* mice. (A) 9V;C*, 0S;C* and 0S pups had ichthyotic skin compared to smooth skin of WT and WT;C* pups. (B) H&E stained skin epidermal sections from 1-day old pups. Normal stratum corneum (SC) in WT (A) and WT;C* (B) skin had basket weave appearance. SC was compact in 9V;C* (C), 0S;C* (D), Gba1−/− (E) and 0S (F) skin epidermis. (C) Ultrastructural studies of stratum corneum in 1-day old pup skin. Normal lamellar structure (arrow) in WT skin. 9V;C* and 0S;C* had loosely packed layers and irregular lamella structure (arrow).

Figure 5

GCase activity. (A) GCase activity in 9H;C* mice tissues were reduced compared to WT and WT;C* mice. Compared to 9H mice, GCase activity in 9H;C* tissues were not changed in the lung, spleen and cerebrum, but slightly elevated in the liver. (B) 9V;C* and 0S;C* mice had reduced GCase activity in the liver, lung and brain compared to 9V and 0S tissues, respectively. Student’s t-test (n = 3–6 mice).

Figure 6

Tissue GC and GS analysis by LC/MS. (A) 9V;C* and 0S;C* mice had GC accumulation in liver and lung. 9V;C* had GC accumulation and 0S;C* mice had WT GC level in brain. (B) 9H;C* visceral and brain tissues showed GC accumulation increased with age. (C) GC levels were increased in 4L;C* brain and lung compared to WT mice. (D) Epidermal GC and GS levels were significantly increased in 9V;C*, 9V, 0S;C* and 0S mice at 1 day of age compared to WT. Epidermal GC and GS levels in 9V;C* were higher than 9V. (E) Total ceramides were significantly increased in 0S, 0S;C*, 9V;C* and WT;C* mice epidermis compared to WT. Epidermal ceramide levels in 9Vmice were not significantly different from WT. Student’s t-test (n = 3–6 mice).