A novel mouse model of a patient mucolipidosis II mutation recapitulates disease pathology

Abstract

Mucolipidosis II (MLII) is a lysosomal storage disorder caused by loss of N-acetylglucosamine-1-phosphotransferase, which tags lysosomal enzymes with a mannose 6-phosphate marker for transport to the lysosome. In MLII, the loss of this marker leads to deficiency of multiple enzymes and non-enzymatic proteins in the lysosome, leading to the storage of multiple substrates. Here we present a novel mouse model of MLII homozygous for a patient mutation in the GNPTAB gene. Whereas the current gene knock-out mouse model of MLII lacks some of the characteristic features of the human disease, our novel mouse model more fully recapitulates the human pathology, showing growth retardation, skeletal and facial abnormalities, increased circulating lysosomal enzymatic activities, intracellular lysosomal storage, and reduced life span. Importantly, MLII behavioral deficits are characterized for the first time, including impaired motor function and psychomotor retardation. Histological analysis of the brain revealed progressive neurodegeneration in the cerebellum with severe Purkinje cell loss as the underlying cause of the ataxic gait. In addition, based on the loss of Npc2 (Niemann-Pick type C 2) protein expression in the brain, the mice were treated with 2-hydroxypropyl-β-cyclodextrin, a drug previously reported to rescue Purkinje cell death in a mouse model of Niemann-Pick type C disease. No improvement in brain pathology was observed. This indicates that cerebellar degeneration is not primarily triggered by loss of Npc2 function. This study emphasizes the value of modeling MLII patient mutations to generate clinically relevant mouse mutants to elucidate the pathogenic molecular pathways of MLII and address their amenability to therapy.

Keywords: Ataxia; Drug Action; Lysosomal Storage Disease; Mouse Genetics; Mucolipidosis II; NPC2; Neurodegeneration.

FIGURE 1.

Gnptab is the gene mutated in nym mutant. A, sequencing of the Gnptab locus identified a single nucleotide change resulting in a coding change from a tyrosine residue (TAT) to a premature stop codon (TAA) in the nym (nym/nym) mouse. B, schematic of human GNPTA α/β subunits presenting the location of the nym mutation and its conservation from mice to humans (indicated by an asterisk in nym mutants and for the human (H. sapiens) mutation Y888X). The precursor protein is cleaved between Lys-928 and Asp-929 by the site 1 protease to produce two catalytically active α and β subunits. TM, transmembrane domain; aa, amino acid. This figure is adapted from Ref. . C, semiquantitative RT-PCR analysis (n = 4) reveals that the mRNA is reduced by 75%. Results are expressed as relative levels after normalization for the internal control 18 S. D, intracellular localization of wild-type and mutant Gnpta in HEK 293 cells. Cells were fixed and stained with monoclonal antibodies against the Myc tag (green), the cis-Golgi marker protein GM130 (red), or the ER marker protein, protein-disulfide isomerase (PDI; red). In merged images, yellow indicates colocalization. Scale bars, 15 μm. Values are expressed as mean S.E. (error bars) (n = 8; *, p < 0.05; **, p < 0.01).

FIGURE 2.

Growth retardation, facial dysmorphism, and reduced life span. A, 1-month-old nym (nym/nym) mice show reduced body size and skeletal abnormalities (e.g. facial dysmorphism and curvature of the spine) in comparison with wild-type (+/+) littermates. B, body weight progression of nym mice is reduced compared with wild-type littermates. C, facial phenotype progresses with age. Particularly evident is the thickening of the eyelids. D, whole body exotic dorso-ventral view of the 12-month-old nym mouse (left) and whole body exotic lateral view of the 12-month-old nym mouse (right). White arrows, abnormal curvature (left) and hunched back (right). E, Kaplan-Meier analysis of wild-type and nym mice (+/+, n = 52; nym/nym, n = 33). Values are expressed as mean ± S.E. (error bars) (n = 8; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

FIGURE 3.

Increased lysosomal enzyme activity in blood sera and inclusion bodies present in mouse fibroblasts, secretory and connective tissue. A, the relative enzymatic activities of the lysosomal hydrolases β-Hexosaminidase (β-Hex), β-mannosidase (β-Man), α-mannosidase (α-Man), β-glucuronidase (β-Glu), β-galactosidase (β-Gal), α-galactosidase (α-Gal), and β-glucocerebrosidase (β-GCase) were measured in blood sera of 3-month-old wild-type (+/+) and nym (nym/nym) mice. The specific activities of the wild-type were set to 1. Values are expressed as mean ± S.E. (error bars) (n = 8; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). B, light microscopy imaging of MEFs isolated from wild-type and nym embryos (E12.5). Accumulation of inclusion bodies present in the nym MEFs. Scale bar, 40 μm. C, top, the cytoplasm of hypertrophic chondrocytes is distended by microvacuoles in the nym mouse. These inclusions are aggregates of polysaccharides that increase in storage material with age. Bottom, marked disorganization of the pancreas in the nym mouse with tightly packed cells distended by large vacuoles. Scale bar, 50 μm.

FIGURE 4.

The nym heterozygote mouse shows no obvious pathology except for an increase in enzymatic activity similar to carriers of mucolipidosis II. A, the relative enzymatic activities of the lysosomal hydrolases β-Hexosaminidase (β-Hex), β-mannosidase (β-Man), α-mannosidase (α-Man), β-glucuronidase (β-Glu), β-galactosidase (β-Gal), α-Galactosidase (α-Gal), and β-glucocerebrosidase (β-GCase) were measured in blood sera of 3-month-old wild-type (+/+) and nym/+ mice. The specific activities of the wild type were set to 1. Three-month-old heterozygote (nym/+) mice are similar in size to the wild-type (+/+) littermates (B), and body weight progression of nym/+ mice is not significantly different from that of wild-type mice (C). D, facial phenotype of the nym/+ and wild-type mouse are no different. E, Kaplan-Meier analysis of wild-type and nym/+ mice (+/+, n = 52; nym/+, n = 82). nym/+ mice present with the same motor coordination on the rotarod (F) and muscle strength (G), which was measured on the inverted screen, as the wild-type littermate. Values are expressed as mean ± S.E. (error bars) (n = 8; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

FIGURE 5.

Impairments in motor and cognitive function. nym (nym/nym) mice present with reduced motor coordination on the rotarod (A) and reduced muscle strength (B), which was measured on the inverted screen. C, furthermore, gait analysis was conducted via the Noldus Catwalk system, in which most prominently the regularity index was significantly reduced and progressed with age in the nym mouse in comparison with the wild-type (+/+) control. D, the regularity index was not significant at 3 months; however, the base of support (BOS) for the front paws was significantly increased, and this indicates that the front paws are wider apart than in the wild-type control, giving more stability. E, percentage of spontaneous alternation in the Y-maze. Values are expressed as mean ± S.E. (error bars) (n = 8; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 6.

Pathological alterations in the cerebellum. A, the nym (nym/nym) brain is about 30% smaller than the wild-type (+/+) brain. Scale bar, 3 mm. B, parasagittal brain sections were immunostained for calbindin. Analysis of PC degeneration in the whole cerebellum in 3-, 7-, and 11-month-old nym mice showed progressive PC loss. Lobe IV/V is shown as a representative lobe of the cerebellum. The majority of Purkinje cells in lobe IV/V are still present at 3 months, but only 75% remain at 7 months, and only 40–50% remain at 11 months. Scale bar, 100 μm. C, higher magnification images of the 3-month-old nym brain revealed that axonal spheroids/torpedoes precede Purkinje cell loss. Swelling of Purkinje cell axons (arrow) and neuronal torpedoes (*) in the white matter are observed already at 3 months and increase in severity with age. Scale bars, 50 μm. D, luxol fast blue staining of the cerebellar sections indicates decreased myelination and degeneration of the subcortical white matter in the nym mouse. Higher magnification images are shown below in panels 1–4 (1 and 3, +/+; 2 and 4, nym/nym). Scale bars, 200 μm (top) and 100 μm (bottom). E, strong immunoreactivity for glial fibrillary acidic protein (GFAP) in the cerebellum of the nym mouse brain. Scale bar, 50 μm.

FIGURE 7.

Biochemical alterations in the brain. A, the relative enzyme activities of the lysosomal hydrolases β-hexosaminidase (β-Hex), β-mannosidase (β-Man), α-mannosidase (α-Man), β-glucuronidase (β-Glu), β-galactosidase (β-Gal), α-galactosidase (α-Gal), and β-glucocerebrosidase (β-GCase) were measured in whole brain homogenates of wild-type (+/+) and nym (nym/nym) mice (4 months of age). The specific activities of the wild type were set to 1. Values are expressed as mean ± S.E. (error bars) (n = 4; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). B, representative Western blot showing levels of Npc2 in wild-type and nym mice. β-Tubulin was used as a loading control (n = 4). C, nine-month-old nym and wild-type brain sections were stained with filipin for the detection of cholesterol. The molecular layer of the nym cerebellum showed increased storage in comparison with the wild-type control. Scale bar, 50 μm. D, nine-month-old nym and wild-type brain sections were stained with periodic acid-Schiff for detection of glycolipids. Increased storage was detected in the cerebellum (top, specifically in the molecular layer), the cortex (middle, specifically in layers 3–5), and the hippocampus (bottom, specifically in layers CA1 and CA3). Scale bar, 50 μm.

FIGURE 8.

Cyclodextrin does not delay cerebellar pathology and motor impairments. P7 nym (nym/nym) mice were injected weekly for 7 months with cyclodextrin (CD) at 4000 mg/kg or the vehicle PBS (n = 7 in each group). A, no PC loss was observed at 3 months, as can be seen in lobe IV/V as a representative lobe of the cerebellum. Scale bar, 100 μm. B, typical signs of PC degeneration, including swelling of PC dendrites (arrow) and axonal torpedoes (*), can be observed. Scale bars, 50 μm. C, PC loss was observed at 7 months in lobe IV/V as a representation of a lobe of the cerebellum in nym PBS- and nym CD-treated mice. Scale bar, 100 μm. D, typical signs of PC degeneration, including swelling of PC dendrites (arrow) and axonal torpedoes (*), can be observed. Scale bars, 50 μm. E–H, 3- and 7-month-old cyclodextrin (CD) treated nym mice perform the same as PBS and untreated nym mice on the inverted screen (3 months (E) and 7 months (G)) and rotarod (3 months (F) and 7 months (H)). Values are expressed as mean ± S.E. (error bars) (n = 8). +/+, wild-type mice.