Abstract
Deficiencies in the lysosomal hydrolase β-galactosidase (β-gal) lead to two distinct diseases: the skeletal disease Morquio syndrome type B, and the neurodegenerative disease GM1-gangliosidosis. Utilizing CRISPR-Cas9 genome editing, the mouse β-gal encoding gene, Glb1, was targeted to generate both models of β-gal deficiency in a single experiment. For Morquio syndrome type B, the common human missense mutation W273L (position 274 in mice) was introduced into the Glb1 gene (Glb1W274L), while for GM1-gangliosidosis, a 20 bp mutation was generated to remove the catalytic nucleophile of β-gal (β-gal−/−). Glb1W274L mice showed a significant reduction in β-gal enzyme activity (8.4–13.3% of wildtype), but displayed no marked phenotype after one year. In contrast, β-gal−/− mice were devoid of β-gal enzyme activity (≤1% of wildtype), resulting in ganglioside accumulation and severe cellular vacuolation throughout the central nervous system (CNS). β-gal−/− mice also displayed severe neuromotor and neurocognitive dysfunction, and as the disease progressed, the mice became emaciated and succumbed to the disease by 10 months of age. Overall, in addition to generating a novel murine model that phenotypically resembles GM1-gangliosidosis, the first model of β-galactosidase deficiency with residual enzyme activity has been developed.
Highlights
When a lysosomal hydrolase is defective, a single lysosomal disease arises; in the case of the enzyme β-galactosidase (β-gal, EC 3.2.1.23), mutations in the encoding gene, GLB1, can result in two lysosomal diseases: GM1-gangliosidosis (OMIM #230500, 230600, 230650), a neurodegenerative disease, and Morquio syndrome type B (Mucopolysaccharidosis IV B, MPS IVB OMIM #253010), a skeletal disease. β-gal is responsible for catabolizing the terminal galactose residue in GM1 ganglioside, its asialo derivative GA1, and the glycosaminoglycan keratan sulfate [1,2]
Β-gal enzyme activity is nominal compared to controls, which results in accumulation of GM1 and GA1 gangliosides
Zygotes for microinjection were produced by super-ovulating C57BL/6J females by injection of pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) and mated with C57BL/6J stud males. 114 one-cell embryos were collected from the ampulla oviducts the morning after mating and microinjected with 400 nM Cas9 protein, 50 ng/μl guide RNAs (gRNA) targeting exon 8 of Glb1, and 50 ng/μl donor oligonucleotide
Summary
When a lysosomal hydrolase is defective, a single lysosomal disease arises; in the case of the enzyme β-galactosidase (β-gal, EC 3.2.1.23), mutations in the encoding gene, GLB1, can result in two lysosomal diseases: GM1-gangliosidosis (OMIM #230500, 230600, 230650), a neurodegenerative disease, and Morquio syndrome type B (Mucopolysaccharidosis IV B, MPS IVB OMIM #253010), a skeletal disease. β-gal is responsible for catabolizing the terminal galactose residue in GM1 ganglioside, its asialo derivative GA1, and the glycosaminoglycan keratan sulfate [1,2]. GM1-gangliosidosis patients are categorized into one of three disease severities, 1) infantile, 2) late infantile/juvenile, and 3) adult, depending upon symptoms and residual β-gal enzyme activity. In Morquio syndrome type B, patients have residual β-gal enzyme activity In the case of GM1-gangliosidosis, two previously described murine models have been developed [11,12], but were not publicly available when this project was initiated; while no animal models of Morquio syndrome type B exist. Matsuda et al [12] targeted exon 15 In both knockout models, β-gal enzyme activity is nominal compared to controls, which results in accumulation of GM1 and GA1 gangliosides. While mice homozygous for a common Morquio syndrome type B mutation (Glb1W274L) had a present, but significantly reduced, β-gal enzyme activity, Glb1W274L mice displayed no observable Morquio syndrome type B phenotype up to one year of age
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