Proc. Natl. Acad. Sci. USA Vol. 94, pp. 8138–8143, July 1997 Medical Sciences Mouse model of GM2 activator deficiency manifests cerebellar pathology and motor impairment (animal modelyGM2 gangliosidosisygene targetingylysosomal storage disease) YUJING LIU*, ALEXANDER HOFFMANN†,ALEXANDER GRINBERG‡,HEINER WESTPHAL‡,MICHAEL P. MCDONALD§, KATHERINE M. MILLER§,JACQUELINE N. CRAWLEY§,KONRAD SANDHOFF†,KINUKO SUZUKI¶, AND RICHARD L. PROIA* *Section on Biochemical Genetics, Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, ‡Laboratory of Mammalian Genes and Development, National Institute of Child Health and Development, and §Section on Behavioral Neuropharmacology, Experimental Therapeutics Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892; †Institut fu¨r Oganische Chemie und Biochemie der Universita¨tBonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany; and ¶Department of Pathology and Laboratory Medicine, and Neuroscience Center, University of North Carolina, Chapel Hill, NC 27599 Communicated by Stuart A. Kornfeld, Washington University School of Medicine, St. Louis, MO, May 12, 1997 (received for review March 21, 1997) ABSTRACT The GM2 activator deficiency (also known as disorder, the respective genetic lesion results in impairment of the AB variant), Tay–Sachs disease, and Sandhoff disease are the the degradation of GM2 ganglioside and related substrates. major forms of the GM2 gangliosidoses, disorders caused by In humans, in vivo GM2 ganglioside degradation requires the defective degradation of GM2 ganglioside. Tay–Sachs and Sand- GM2 activator protein to form a complex with GM2 ganglioside. hoff diseases are caused by mutations in the genes (HEXA and b-Hexosaminidase A then is able to interact with the activator- HEXB) encoding the subunits of b-hexosaminidase A. The GM2 ganglioside complex and remove the terminal N-acetylgalac- activator deficiency is caused by mutations in the GM2A gene tosamine residue from the oligosaccharide portion of the gangli- encoding the GM2 activator protein. For degradation of GM2 oside. In both Tay–Sachs and Sandhoff diseases, ganglioside ganglioside by b-hexosamindase A, the GM2 activator protein degradation is impaired due to an absence of b-hexosaminidase must participate by forming a soluble complex with the gangli- A. The homodimeric b-hexosaminidase isozymes, S and B, oside. In each of the disorders, GM2 ganglioside and related lipids cannot effectively substitute for b-hexosaminidase A in ganglio- accumulate to pathologic levels in neuronal lysosomes, resulting side degradation. It is believed that the function of the activator in clinically similar disorders with an onset in the first year of life, protein is to transform the hydrophobic, membrane-bound gan- progressive neurodegeneration, and death by early childhood. glioside into a substrate complex that is accessible to the water- We previously have described mouse models of Tay–Sachs (Hexa soluble enzyme (2). Therefore, in the GM2 activator deficiency, 2y2) and Sandhoff (Hexb 2y2) diseases with vastly different the ganglioside is refractory to enzymatic hydrolysis by b-hex- clinical phenotypes. The Hexa 2y2 mice were asymptomatic osaminidase A. In each disease, a massive accumulation of GM2 whereas the Hexb 2y2 mice were severely affected. Through gene ganglioside and related lipids occurs in neuronal lysosomes, disruption in embryonic stem cells we now have established a resulting in severe cellular malfunction and damage. The clinical mouse model of the GM2 activator deficiency that manifests an phenotype of each disease is nearly identical. In the most severe intermediate phenotype. The Gm2a 2y2 mice demonstrated forms, the onset of the disease occurs during the first year of life. neuronal storage but only in restricted regions of the brain Rapidly progressing neurodegeneration culminates in demise (piriform, entorhinal cortex, amygdala, and hypothalamic nu- usually by the age of 4. Disorders of lesser severity and later-onset clei) reminiscent of the asymptomatic Tay–Sachs model mice. result when mutations lead to a partial deficiency in ganglioside However, unlike the Tay–Sachs mice, the Gm2a 2y2 mice degradation capacity. displayed significant storage in the cerebellum and defects in Through targeted gene disruption of the Hexa and Hexb balance and coordination. The abnormal ganglioside storage in genes in mouse embryonic stem (ES) cells, we previously have the Gm2a 2y2 mice consisted of GM2 with a low amount of GA2. described the creation of mouse models corresponding to The results demonstrate that the activator protein is required for Tay–Sachs and Sandhoff diseases (3–5). Unlike the human GM2 degradation and also may indicate a role for the GM2 diseases, the two mouse models were of dramatically different activator in GA2 degradation. phenotypes as a result of differences in the ganglioside deg- radation pathway between mice and humans. The Tay–Sachs The G gangliosidoses are severe neurodegenerative disor- model developed ganglioside accumulation in restricted re- M2 gions of the brain but was asymptomatic. The Sandhoff disease ders caused by excessive accumulation of GM2 ganglioside. Three genetic forms exist: Tay–Sachs disease, Sandhoff dis- model exhibited much more extensive ganglioside accumula- tion and developed a severe neurologic phenotype. ease, and the GM2 activator deficiency (also known as the AB variant) (reviewed in ref. 1). Tay–Sachs disease is caused by We now have established mice with a disrupted Gm2a gene as mutations in the HEXA gene, Sandhoff disease by mutations a model for the third form of the GM2 gangliosidoses. Similar to the Hexa 2y2 mice, the Gm2a 2y2 mice demonstrated storage in the HEXB gene, and the GM2 activator deficiency by mutations in the GM2A gene. The HEXA and HEXB genes in restricted regions of the brain. However, these mice also encode the a- and b-subunits, respectively, of the lysosomal displayed some significant differences. They exhibited storage in Hexa enzyme, b-hexosaminidase. The subunits dimerize to form the the cerebellum, a site of minimal pathology in the 2y2 mice (4) and developed defects in balance and motor coordination. three b-hexosaminidase isozymes, A (ab), B(bb), and S (aa). This mouse model of the G activator deficiency is, thus, of an The GM2A gene encodes the G activator protein. In each M2 M2 intermediate phenotype when compared with the severely af- The publication costs of this article were defrayed in part by page charge Abbreviations: ES, embryonic stem; G ganglioside, Gal- payment. This article must therefore be hereby marked ‘‘advertisement’’ in M1 b133GalNAcb134(NeuAca233)Galb134Glcb131Cer; G gangli- accordance with 18 U.S.C. §1734 solely to indicate this fact. M2 oside, GalNAcb134(NeuAca233)Galb134Glcb131Cer; GA2 glyco- © 1997 by The National Academy of Sciences 0027-8424y97y948138-6$2.00y0 lipid, GalNAcb134Galb134Glcb131Cer; GM3 ganglioside, NeuAc- PNAS is available online at http:yywww.pnas.org. a233Galb134Glcb131Cer; Cer, ceramide. 8138 Downloaded by guest on September 25, 2021 Medical Sciences: Liu et al. Proc. Natl. Acad. Sci. USA 94 (1997) 8139 fected Sandhoff model and the asymptomatic Tay–Sachs model. offspring were tested for transmission of the disrupted allele by The basis for the distinct phenotypes of the human and mouse Southern blot analysis. Heterozygous matings were set up to GM2 gangliosidoses are species-specific differences in the gangli- generate homozygous mutant mice. oside degradation pathway. Biochemical Analysis. b-Hexosaminidase assays were per- formed as described (3). Methodology for the analysis of brain MATERIALS AND METHODS lipids from gray and white matter has been described (5). Targeting Vector Construction. We previously have con- Pathology. Six Gm2a 2y2 and two Gm2a 1y2 mice, ages structed a plasmid containing both the neomycin-resistance ranging from 2 to 7 months, were studied. Mice were anesthetized (neo) gene and the thymidine kinase gene in the pBluescript and perfused with phosphate-buffered 4% paraformaldehyde KS vector (3). Genomic clones from a 129ySV strain library (pH 7.4). The brain, spinal cord, and pieces of visceral organs (Stratagene; catalogue no. 946306) containing the mouse from two of the Gm2a 2y2 mice were processed for paraffin Gm2a gene were isolated and characterized. A 4.5-kb genomic sections, cut and stained with hematoxylin-eosin, solochrome- fragment containing part of intron 2 was inserted between the eosin, Luxol-fast blue-periodic acid-Schiff, and Bielschowsky SalI and NotI sites downstream of the Neo gene. Next, a 4.5-kb stains. In other mice the cerebrum, cerebellum, brainstem, and spinal cord were cryoprotected by immersion in 0.1 M phosphate genomic fragment containing part of the exon 4 and down- buffer (pH 7.4) containing 30% sucrose, snap-frozen in a Hist stream region was cloned into the XhoI site between the Bath (Shandon, Pittsburgh), and serially sectioned at 7 mm thick thymidine kinase and Neo genes. The organization of the with a Frigocut 2800 (Leica, Germany). Every fourth section was targeting vector is illustrated in Fig. 1A. The design of the stained with periodic acid-Schiff and counter-stained lightly with targeting vector would result in a 1-kb deletion that includes hematoxylin. the entire exon 3, entire intron 3, and part of exon 4. Behavioral Methods. Subjects were 24 mice (six wild type, six Selection of Targeted ES Cells and Generation of Mutant Gm2a 1y2, and 12 Gm2a 2y2). All procedures were approved Mice. The targeting vector (50 mg) was linearized with SspI by the National Institute of Mental Health Animal Care and Use endonuclease and introduced into the J1 line (6) of ES cells (1.6 3 Committee and followed the National Institutes of Health guide- 7 10 cells) by electroporation (400 V and 25 mF) in a Bio-Rad lines, ‘‘Using Animals in Intramural Research.’’ Mice were 13 Gene Pulser. Targeted clones were isolated as described (3). weeks old at the start of testing. Body weights were measured Targeted ES cells lines Act-4 and Act-25 were injected into the biweekly during the 20-week testing period. There were no blastocysts of 3.5-day C57BLy6 embryos.