Glucocerebrosidase gene-deficient mouse recapitulates Gaucher disease displaying cellular and molecular dysregulation beyond the macrophage

Abstract

In nonneuronopathic type 1 Gaucher disease (GD1), mutations in the glucocerebrosidase gene (GBA1) gene result in glucocerebrosidase deficiency and the accumulation of its substrate, glucocerebroside (GL-1), in the lysosomes of mononuclear phagocytes. This prevailing macrophage-centric view, however, does not explain emerging aspects of the disease, including malignancy, autoimmune disease, Parkinson disease, and osteoporosis. We conditionally deleted the GBA1 gene in hematopoietic and mesenchymal cell lineages using an Mx1 promoter. Although this mouse fully recapitulated human GD1, cytokine measurements, microarray analysis, and cellular immunophenotyping together revealed widespread dysfunction not only of macrophages, but also of thymic T cells, dendritic cells, and osteoblasts. The severe osteoporosis was caused by a defect in osteoblastic bone formation arising from an inhibitory effect of the accumulated lipids LysoGL-1 and GL-1 on protein kinase C. This study provides direct evidence for the involvement in GD1 of multiple cell lineages, suggesting that cells other than macrophages may be worthwhile therapeutic targets.

Fig. 1.

GBA1 mice display a visceral and hematologic phenotype. (A) Gross appearance of a 14-mo-old GBA1 mouse showing severe runting, Gibbus formation, and striking extremity pallor. (B) Hepatomegaly and (C) splenomegaly in GBA1 mice demonstrate areas of prominent surface infarction; note the difference in texture and color of the GBA1 organs. (D) Comparison of (i) spleen weight and (ii) liver weight as a percentage of body weight, as well as (iii) white blood cell (WBC) count, (iv) hemoglobin (Hb), and (v) platelet (PLT) count in GBA1 compared with control mice. Note the anemia and reduced platelet count. Student t test, **P < 0. 01, control versus GBA1, n = 19–59 mice/group. (E) H&E staining showing focal collections of classical foamy Gaucher cells (arrows) in two representative sections of liver, spleen, lymph nodes, lungs, and thymus of GBA1 mice. Control (WT) sections are shown. (E, Lower). Extensive extramedullary hematopoiesis in liver and spleens of GBA1 mice; arrows denote megakaryocytes. (F) The extensive accumulation of tubular storage material within liver Gaucher cells was confirmed on transmission EM.

Fig. 2.

Marked alternations in immune cell populations in the GBA1 mouse thymus. Thymocytes were isolated from 6-mo-old control (WT) and GBA1 mice and labeled with antibodies to CD4, CD8, CD11b, B220, TCRα/β, CD44, CD69, IA/IE, CD11c, NK1.1, and CD25 for flow cytometry (Materials and Methods). Severely diseased GBA1 mice, selected as having a spleen size greater than five times the normal size, but having normal thymic weight and cellularity, were analyzed. Shown are representative data from three separate experiments.

Fig. 3.

Bone marrow infiltration, avascular necrosis, and systemic osteopenia in GBA1 mice. (A) Control vertebral section with adjacent bone marrow (i) shows marked differences fromGBA1 mice (ii), which have focal collections of Gaucher cells (arrow) interspersed within the normal vertebral hematopoietic marrow. (iii) GBA1 mouse bone section showing medullary infarction and avascular necrosis; also shown is an extrusion of Gaucher cells into skeletal muscle. Control section (iv) of femoral cortical bone compared with a GBA1 section (v) showing disorganized osteocyte lacunae with a loss of lamellar structure. (B) Areal BMD (aBMD) using a Lunar Piximus densitometer in 14-mo-old GBA1 and control (WT) mice at six sites, namely lumbar spine (L4–L6 and >L6) and right and left femurs and tibiae as shown. Student t test was used to compare GD1 and WT mice; n = 2–10 mice/group; *P < 0. 05, **P < 0. 01. (C and D) Micro-CT of lumbar spine shows representative images of lumbar vertebrae, as well as the mean and range (parentheses) of BV/TV, trabecular number (Tb.N), trabecular thickness (Tb.Th.), and trabecular spacing (Tb.Sp.) in GBA1 mice compared with WT littermates (n = 2–10 mice/group). The orange color in the composite images shows a reduction in mineralized bone (D). MTT assay (E) shows proliferation of bone marrow stromal cells from WT and GBA1 mice with or without PMA. (F and G) ALP-positive cfu-f and (H) von Kossa-positive cfu-ob from WT and GBA1 mice with or without PMA. Results are shown as optical density (OD) (E), or cfu-f (%); mean ± SEM, or representative wells. Statistics by Student t test: **P < 0. 01; four mice per group, triplicate estimations for all three experiments. (I) Markers of osteoblast differentiation, namely ALP, BSP, Runx2, and osterix by quantitative PCR in GBA1 mice compared with WT. Student t test was used to compare WT and GBA1 mice; n = 2–10 mice in triplicate wells; *P < 0. 05, **P < 0. 01. (J) Ex vivo TRAP-positive osteoclast formation in WT and GBA1 mice. Student t test was used to compare WT and GBA1; n = 2–10 mice per group, eight wells per group; P > 0.5.

Fig. 4.

LysoGL-1 inhibits osteoblastogenesis via PKC. (A) Proliferation of bone marrow stromal cells in the presence of PMA (100 nM) with or without LysoGL-1 (10 μM) or GL-1 (40 μM), using the MTT assay. (B and C) ALP-positive cfu-f (B) or cfu-ob (C) with or without PMA and LysoGL-1 or GL-1. Results are shown as optical density (OD) (A) or representative wells (B and C). (D) Markers of osteoblast differentiation, namely BSP and Runx2, for 10- and 21-d cultures by quantitative PCR in the presence of PMA with or without Lyso-GL1 or GL-1. Statistics by Student t test: **P < 0. 01 vs. zero dose; n = 4 mice per group, triplicate estimations for all experiments. (E) Differentiation ofMC3T3. E1 osteoblast precursors, assessed by cfu-f formation and quantitative PCR for ALP and Runx 2 (at day 10) with or without PMA and LysoGL-1 or GL-1. Results are shown as representative wells or relative expression. Statistics by Student t test: **P < 0. 01 vs. zero dose, triplicate estimations.