Neuronopathic Gaucher disease in the mouse: viable combined selective saposin C deficiency and mutant glucocerebrosidase (V394L) mice with glucosylsphingosine and glucosylceramide accumulation and progressive neurological deficits

Abstract

Gaucher disease is caused by defective acid beta-glucosidase (GCase) function. Saposin C is a lysosomal protein needed for optimal GCase activity. To test the in vivo effects of saposin C on GCase, saposin C deficient mice (C-/-) were backcrossed to point mutated GCase (V394L/V394L) mice. The resultant mice (4L;C*) began to exhibit CNS abnormalities approximately 30 days: first as hindlimb paresis, then progressive tremor and ataxia. Death occurred approximately 48 days due to neurological deficits. Axonal degeneration was evident in brain stem, spinal cord and white matter of cerebellum accompanied by increasing infiltration of the brain stem, cortex and thalamus by CD68 positive microglial cells and activation of astrocytes. Electron microscopy showed inclusion bodies in neuronal processes and degenerating cells. Accumulation of p62 and Lamp2 were prominent in the brain suggesting the impairment of autophagosome/lysosome function. This phenotype was different from either V394L/V394L or C-/- alone. Relative to V394L/V394L mice, 4L;C* mice had diminished GCase protein and activity. Marked increases (20- to 30-fold) of glucosylsphingosine (GS) and moderate elevation (1.5- to 3-fold) of glucosylceramide (GC) were in 4L;C* brains. Visceral tissues had increases of GS and GC, but no storage cells were found. Neuronal cells in thick hippocampal slices from 4L;C* mice had significantly attenuated long-term potentiation, presumably resulting from substrate accumulation. The 4L;C* mouse mimics the CNS phenotype and biochemistry of some type 3 (neuronopathic) variants of Gaucher disease and is a unique model suitable for testing pharmacological chaperone and substrate reduction therapies, and investigating the mechanisms of neuronopathic Gaucher disease.

Figure 1.

Phenotypes and lifespan of 4L;C* mice. (A) 4L;C* mouse showed hindlimb paresis and duck waddling by 47 days. They weighed 25% less than control mice. (B) Kaplan–Meier survival curves for 4L;C* and control (WT and C+/−) mice. Average lifespan of 4L;C* mice was 48 days.

Figure 2.

Electrophysiology analyses of long-term potentiation (LTP). The slope of resulting EPSPs from the parasagittal sections (350 µm) of hippocampal CA1 region was recorded. The slopes of the EPSP (LTP) in 35 days 4L;C* (black circle) was significantly (P < 0.05) decreased compared with WT (open circle) mice after recording for 90 min following stimulation. WT, n = 6 mice; 4L;C*, n = 8 mice.

Figure 3.

Neuropathology in 4L;C* mice. The sections from 46-day 4L;C* mice were stained with H&E. Axonal degeneration (arrows) was present in brain stem, spinal cord and white matter of cerebellum in 4L;C* mice. WT brain stem, spinal cord and cerebellum had normal morphology.

Figure 4.

Neuroinflammation in 4L;C* mice. (A) CD 68 staining (brown) in 46-day 4L;C* and WT mice. [A1]WT brain stem had background levels of CD68 staining. CD68 positive staining demonstrated activation of macrophage/microglial cells in 4L;C* brain stem [A2], spinal cord [A3] and thalamus [A4]. Methyl green (green) was used to stain cell nuclei. (B) GFAP staining (green) in 46 days 4L;C* and WT mice. [B1] WT brain stem had background level GFAP staining. Enhanced GFAP staining presented in 4L;C* brain stem [B2], thalamus [B3] and cerebellum [B4]. Cell nuclei were stained with DAPI (blue).

Figure 5.

Accumulation of p62 and Lamp2 in 4L;C* thalamus. (A) Frozen sections from 46-day 4L;C* and WT mice were stained with anti-p62 and anti-Lamp2 antibodies, respectively. Both p62 (green) and Lamp2 signals (green) were increased in thalamus of 4L;C* brain relative to the WT control. (B) Colocalization of p62 in neuron, astrocyte or microglial cells in the thalamus of 4L;C* brain. Accumulation of p62 (red) was in neuron (green) stained by anti-NeuN antibody and astrocyte (green) stained with anti-GFAP antibody. Arrows point the p62 containing cells. Yellow color cell shows the overlapping of p62 and GFAP or NeuN. p62 (red) was not localized in CD 68 positive microglial cells (green). DAPI (blue) in anti-fade was used for cell nuclei staining.

Figure 6.

Ultrastructural features of 4L;C* CNS. (A) Normal midbrain morphology in 43 day WT. (B) Axonal inclusions (arrows) were in 43-day 4L;C*. (C) 4L;C* spinal cord showed storage materials in axonal process (arrow) and separation of myelin layers (arrowhead). (D) Brain stem of 4L;C* had normal neuron. (E) The white matter of cerebellum in 4L;C* mice contained a complex accumulation materials in axonal process. (F) Higher magnification of the complex accumulation in cerebellar axons showed amorphous granular inclusions of variable density. The storage materials are heterogeneous and coarsely granular.

Figure 7.

GCase activity and protein in 4L;C* mice. (A) The activities of GCase decreased significantly in 4L;C* liver, brain, lung, spleen and fibroblasts compared with 4L/4L, C−/− and WT tissues (n = 3). (B) GCase protein was decreased in 4L;C* livers by immunoblot analyses using anti-mouse GCase antibody. GCase protein level was normalized to β-actin signals in the same sample and presented as ratio relative to 4L/4L. A representative immunoblot is shown. The data represented the means ± SE for three mice assayed in duplicates. **P < 0.01; ***P < 0.001.

Figure 8.

LC/MS analyses of GC and GS. (A) GS levels showed marked increases (20- to 600-fold) by 43 days in 4L;C* cortex, midbrain, liver and lung. (B) Increases of GC (1.5- to 3-fold) were detected in 4L;C cortex, midbrain, liver and lung. These GC and GS levels were normalized to phosphate content in the same sample and presented as fold change relative to WT controls. (C) Both GC (3.4-fold) and GS (132-fold) levels were significantly increased in 4L;C* hippocampus relative to WT. GalCer in 4L;C* hippocampus was at WT level. The hippocampal GC, GS and GalCer levels were normalized to mg protein in the same sample. The data were analyzed by Student's t-test. *P < 0.05; **P < 0.01, ***P < 0.001 (n = 3 mice).