Vet. Pathol. 16: 635-649 (1979) The Gangliosidoses: Comparative Features and Research Applications H.J. BAKER,G. D. REYNOLDS, S. U. WALKLEY, N. R. Cox and G. H. BAKER Department of Comparative Medicine, Schools of Medicine and Dentistry, University of Alabama in Birmingham, Birmingham, Ala. Abstract. Ganglioside storage diseases are inherited defects of lysosomal hydrolases that result in intralysosomal accumulation of gangliosides and other complex metabolites. Gangliosidoses occur in man, cats, cattle, dogs and swine. In all species, these diseases are characterized clinically by relentlessly progressive neurological deterioration. Lysosomal hypertrophy with characteristic ultrastructural inclusions occur in neurons, endothelial and other cells. Definitive diagnosis requires biochemical identification of the storage product and enzyme deficiency. Gangliosidoses in animals are important models of human lysosomal diseases and may be a significant complication in the maintenance of certain purebred stocks of domestic animals. According to current concepts, the lysosomal system is the principal site of intracellular digestion and consists of membrane-bound cytoplasmic organelles containing more than 40 acid hydrolases capable of degrading most biologically important macromolecules. Mutations that cause reduced hydrolytic activity of lysosomal hydrolases result in diseases characterized by incomplete catabolism and concomitant accumulation (“storage”) of undergraded substrate within lysosomes [7, 9, 181. The gangliosidoses are lysosomal diseases resulting from incomplete catabolism and intralysosomal accumulation of gangliosides and related complex glycolipids and glycoproteins. These diseases have been recognized in five mammalian species including man, cats, cattle, dogs and swine (31. Regardless of species affected, the gangliosidoses are characterized by 1) progressive nervous system deterioration that usually begins early in life and ultimately leads to premature death; 2) autosomal recessive inheritance; 3) lysosomal hypertrophy in neurons, hepatocytes, macrophages and other cells resulting from deposition of glycoproteins or glycolipids; and 4) absence or marked reduction in activity of specific lysosomal enzymes required for hydrolysis of accumulated compounds. Most lysosomal storage diseases, including the gangliosidoses, are untreatable. Furthermore, the pathogenesis of cell injury and death resulting from lysosomal hypertrophy remains obscure. Therefore, animal models of these diseases are of critical importance for progress in basic and applied research. Also, it is becoming 635 Baker et a1 636 Table 1. Major clinicopathological features of human gangliosidoses GM2 gangliosidoses Type I (TaySachs') Age of onset of symptoms Age at death Mental/motor retardation Facial appearance Edema X-ray changes, long bones X-ray changes, vertebrae Vacuolated lymphocytes Foam cells in marrow Hepa tomegaly Splenomegaly Cherry-red spot Startle response to sound Macrocephaly Macroglossia Seizures Blindness Neuronal lipidosis Visceral histocytosis Glomerular epithelial ballooning Mucopolysakhariduria Type I1 (Sandhoff's disease) 3-6 3-6 months months 2-5 2-5 months months GM I gangliosidoses Type 111 (juvenile) Birth 6-20 months 5- I5 years 1 /2-2 years 3-10 months Coarse Normal - Mild Mild +' + + Doll-like Normal - - - 90% + + + Early + - - + + + - gliosidosis) Type I1 (juvenile) 2-6 years Doll-like - Type I (generalized gan- - - - + - + + + + + + 50% + Rarely + + - + + - - + - Mild - - + + + + + Late + + + - - * f + Early + - + Late + + Early ~ + = present; - = absent. increasingly apparent that gangliosidoses are not uncommon in purebred animals and may constitute a significant complication in the maintenance of some purebred stocks. Comparative Features Following the first clinical description of a human gangliosidosis (Tay-Sachs disease) in 1881, more than 86 years elapsed before a reasonably complete understanding of the basic biochemical defect in these diseases emerged and additional clinical forms of gangliosidoses were recognized. Presently, five clinically distinct human gangliosidoses are well documented [27, 38, 441; the principal differences are outlined in table I. The first documented ganglioside storage disease of domestic animals was a case of GM2 gangliosidoses in German Shorthair Pointer dogs in 1967 . The specific Gangliosidoses 637 Table 11. Ganglioside storage diseases in man and animals GMI gangliosidoses GM2 gangliosidoses Human disease Animal analog’ Generalized gangliosidosis, type 1, Norman-landing disease Juvenile GMI gangliosidosis, type 2, Derry’s disease Bovine GMI gangliosidosis Friesian cattle Feline GMI gangliosidosis Siamese, Korat and mixed Canine GMI gangliosidosis Canine GM:, gangliosidosis Beagle/mixed breed dogs German Shorthaired Pointer dogs Porcine GM2 gangliosidosis Yorkshire swine Feline GM2 gangliosidosis Mixed breed cats GM2 gangliosidosis type 1, Tay-Sachs disease GM2 gangliosidosis type 2, Sandhoff’s disease Juvenile GM2 gangliosidosis, type 3, BernheimerSeitelberger disease Breed ’ Arrangement in table does not imply analogy between animal disease and clinical subtype of human disease. enzyme defect, however, remains undefined. Since 1971, additional cases of gangliosidoses with complete biochemical confirmation have been reported in cats [ 1, 2, 4, 5, 171, cattle [ll, 121, dogs [16, 24, 331 and swine [28, 341 (table 11). Clinical characteristics Although some differences exist in age of onset, severity of signs and rapidity of progression, clinical characteristics are remarkably similar in all species, including man. Relentlessly progressive neurological dysfunction is the sine qua non of these diseases. Discrete head or limb tremors and dysmetria constitute the earliest signs in animals and first become apparent when the animal is 3 months old or older. Locomotor deficits progress in intensity over succeeding months and terminate in quadraplegia, somnolence, blindness and epileptiform seizures. The progressive nature of neurological signs is useful for differentiating the gangliosidoses from other neurological disorders of early onset, such as feline cerebellar hypoplasia. The age of onset of neurological signs in children varies considerably with clinical variants and a few cases are reported in adults with relatively mild neurological deficits . Children with some forms of gangliosidoses have a distinctive retinal lesion, the “cherry red spot,” resulting from lipid filled retinal cells that form a pale ring around the red macula . In animals, retinal lesions have been seen only in swine with GM2gangliosidosis in which punctiform retinal lesions are a consistent, characteristic clinical sign 1281. Corneal clouding has been seen in feline GMI and GM2 gangliosidoses. This 638 Baker era/ apparently results from lysosomal storage of proteoglycans in corneal endothelial cells and fibroblasts [5, 251. Clinically apparent hepatosplenomegaly and skeletal abnormalities, which are prominent features of some human gangliosidoses, have not been seen in other species. Genetics All of the gangliosidoses for which adequate data are available seem to be inherited as autosomal recessive traits. Heterozygotes for these traits are phenotypically normal, but have about half-normal activity of the pivotal lysosomal hydrolase. This unique feature has been used to survey high risk human populations and can be an effective tool for eliminating carrier animals in domestic animal breeding programs or for selective breeding in research colonies. Consanguinity is a consistent feature of the expression of these diseases in domestic animals. Biochemistry The putative catabolic pathway of gangliosides is shown in figure I . The gangliosidoses are classified into two major biochemical subgroups, GMI or GM2 gangliosidoses, based on the nature of the storage product. GM, gangliosidosis results from a block in the hydrolysis of the terminal galactose moiety normally achieved by one or more isozymes of acid optimal B-galactosidases. Similarly, GM2 gangliodosis results from the failure of B-hexosaminidases to cleave hexosamine terminals. Clinical subtypes of human GM2 gangliosidoses correlate with partial or complete defects of hexosamine isozymes. Of the animal analogs of GM2 gangliosidoses, only the feline disease is known to be an exact biochemical replica of human infantile GM2 gangliosidosis (Type I1 or Sandhoff's disease) in which both isozymes are inactive [51. Total ganglioside content of cortical brain tissue in diseased animals is high, reaching levels two to three times normal. Because GM2 ganglioside is normally a minor component of the total ganglioside pool (less than l%), in GM2 gangliosidosis the relative increases in this compound is particularly high. Asialo derivatives (sialic acid free) of the gangliosides also accumulate in brain and liver of most species. High concentrations of other neutral glycosphingolipids are found in some gangliosidoses. Visceral storage of glycolipids and glycoproteins is characteristic of human, feline and canine GMI gangliosidoses, but not bovine GM 1 gangliosidosis . Hepatocellular lysosomal hypertrophy also is found in Sandhoff's disease of man and the feline counterpart [ 6 ] , but not canine or porcine GM2 gangliosidosis. The hepatocellular storage product in GMI gangliosidosis is a large molecular weight glycopeptide with nonreducing galactose in p-D linkage [ 191. These compounds are highly water soluble and are usually leached from tissue fixed with usual aqueous fixatives. In both GMI and GM2gangliosidoses hepatocellular storage includes asialo gangliosides and other glycolipids. Macrophages and endothelial cells often accumulate storage products. 639 Gangliosidoses gal-NAcgal-gal-glc-cer GM 1 Ganglioside GM2 Ganglioside hexororninidase gal-glc-cer GM3 Ganglioside Neurarninidose gal-glc-cer LactosyI ce ramide glc-cer Glucosyl ceramide ' t Ceramide Fig. 1: Sequential catabolic pathway of gangliosides. Lysosomal hydrolase (in box) required for degradation of each compound. cer Morphology Except for muscle atrophy associated with prolonged neurological- disease, gross lesions are absent in animals other than man. Slight macroencephaly and hydrocephalus are associated with some human gangliosidoses but are not found in animals. In the terminal stages of disease the brain may be moderately firm in all species. Lesions in nervous tissue are remarkably consistent, regardless of species affected or biochemical subtype. Lysosomal hypertrophy caused by accumulation of gangliosides is apparent in most neurons even before birth. When clinical signs are advanced, routine histological examination of brain, spinal cord or peripheral ganglia reveals widespread neuronal degeneration characterized by varying degrees of swelling, cytoplasmic vacuolation, loss of Nissl substance, margination of nuclei or loss of neurons (fig. 2). In frozen sections the cytoplasm of affected neurons and glial cells stain intensely with periodic acid-Schiff (PAS) and faintly with stains for neutral fats. Epoxy embedded thick sections (0.5 micrometer) stained with toluidine blue and examined under the light microscope have dense blue, oval to round inclusions that fill the cytoplasm of neurons and glial cells (fig. 3). Ultrastructurally, these inclusions are spherical bodies about 1 micrometer in diameter consisting of multiple concentric Fig. 2 Purkinje cells from the cerebellum of a cat with GMI gangliosidosis. HE. Fig. 3 Cortical neurons of cat with GM I gangliosidosis. Hypertrophied lysosomes. Epoxy embedded. Toluidine blue. Fig. 4 Transmission electron micrograph of membranous inclusion bodies in lysosomes of neuron. Glutaraldehyde and OsO,, lead citrate. 9 Fig. 5 Liver from cat with G M , gangliosidosis. Large unstained vacuoles in cytoplasm of most hepatocytes. HE. 640 Gangliosidoses 64 1 lamellae with an interlamellar periodicity of 500 to 600 nanometers (fig. 4). The fine structure of these inclusions is identical to that of the membranous cytoplasmic bodies typically found in human gangliosidoses. Gliosis and demyelination are significant only in the terminal stages of disease. Recent studies with the rapid Golgi technique have shown bizarre morphological abnormalities, known as meganeurites, in cats  with GMI gangliosidosis and children  with gangliosidoses and Hurler's syndrome. These meganeurites, between the perikaryon and axon, appear to give rise to neurites and dendritic spines. Some of these projections form synapses with presynaptic fibers of unknown origin. Hepatocellular lesions are characteristically found in human ,feline  and canine  GMI gangliosidoses, as well as human  (Sandhoff s disease) and feline  GMPgangliosidosis. Lesions in liver prepared by routine histological procedures consist of diffuse vacuolation representing distended lysosomes from which watersoluble glycopeptidesleached during fixation (fig. 5,6). After glutaraldehyde-osmium fixation, these vesicles can be shown to contain colloidal-iron-positive material, presumed to be a proteoglycan with nonreducing terminal galactose residues (fig. 7). Ultrastructural examination shows lysosomes of hepatocytes and Kupffer cells distended with material that has granular or lamellar structure . Endothelial cells and perivascular macrophages in many organs are vacuolated. Cytoplasmic vacuolation and lysosomal inclusions also have been found in pancreatic acinar cells, renal tubular epithelium, myocardial cells, corneal stroma and cultured fibroblasts . Testes of pubescent cats homozygous recessive for GM I gangliosidosis show a normal complement of spermatogonia, but are virtually devoid of mature spermatozoa. Laboratory diagnosis The appearance in recent years of numerous reports of gangliosidoses in a variety of species indicates the potential importance of these diseases as complications in the maintenance of purebred domestic animal stocks. Current diagnostic technology is sufficiently advanced to permit the detection and elimination of heterozygous carriers from purebred stocks. Furthermore, the value of these disorders as models for research on human lysosomal diseases has been documented  and further development of mutant stocks for research is needed. For these reasons, it is imperative that cases of gangliosidoses be fully investigated and documented. Veterinary clinicians should consider the gangliosidoses in the differential diagnosis of animals with progressive generalized locomotor disease that first appears soon after weaning. In addition to routine data, the clinical record should include thorough documentation of the pedigree, with special reference to occurrences of previous cases in the family, and complete description of neurological signs, including age of onset and rate of progression. A motion picture record of neurological signs is valuable. Accurate laboratory confirmation of suspect cases can be done by morphological and biochemical methods. Sample collection at necropsy should include preservation 642 Baker er al Fig.6 Epoxy embedded thick section of liver from cat with GM, gangliosidosis. Single large vesicle and multiple small vesicles in cytoplasm of hepatocytes and Kupffer cells. Toluidine blue. Fig. 7: Thick section of liver of cat with GMIgangliosidosis.Colloidal-iron-positivematerial in vacuoles of hepatocytes and Kupffer cells. Glutaraldehyde-osmium,colloidal-iron. of generous portions of brain, liver and kidney in air tight containers at -4" C or colder; processing representative parts of brain, spinal cord and visceral organs for routine light and electron microscopy and frozen sectioning; and aseptic excision of skin for fibroblast culture. Tentative diagnoses may be based on the observation of typical lesions in neurons, hepatocytes and macrophages [2, 6, 131. Glycolipid storage in neurons and glia should be confirmed by histochemistry. Demonstration of multilamellar inclusions by electron microscopy completes the morphological assessment. Final diagnosis must be based upon biochemical demonstration of accumulated ganglioside storage product or deficiency of the corresponding lysosomal hydrolase, or both. Assistance in biochemical evaluation of tissues from suspected cases should be sought from laboratories specializing in diagnosis of sphingolipid storage diseases or scientists studying these diseases . Quantitative determination of gangliosides in brain is done by differential solvent extraction [ 141, thin-layer chromatography [411, and quantification of the sialic acid Gangliosidoses 643 Table 111. Enzyme specific activities in cultured fibroblasts Mean P-galactosidase Mean P-hexosaminidase specific activity (Na- specific activity (Nanonomoles cleaved/mg moles cleaved/mg proprotein/hr f SD2 (n)3) tein/hr k SD2 (n)3) Genotype' Normal (dominant) (8) GMI heterozygote (3) GMI homozygous recessive (10) GM2 heterozygote (2) GM2 homozygous recessive (6) 138 f 6 (20) 79 f 6 (12) 4 f 3 (20) 138 f 5 (12) 166 & 20 (20) 3 182 f 282 (20) 3189 & 373 (12) 3482 f 371 (20) 1724 f 93 (12) 101 12 (20) * ' Number in parenthesis = number of cell lines derived from different animals. ' One standard deviation. 'Number in parenthesis = number of samples. content of separated gangliosides . Ganglioside analysis is done best on fresh or frozen brain, but patterns of diagnostic value can be assessed in fixed brain tissue preserved in aqueous buffered formalin for less than a year . Definitive biochemical confirmation of suspected gangliosidoses can be made by assay of tissue for GM ganglioside @-galactosidaseand @-hexosaminidases.Routine diagnostic assay of enzyme activity uses chromogenic or fluorogenic synthetic substrates. Practical methods for assay of GM1 ganglioside @-galactosidaseand @hexosaminidases have been described [39, 421. Homozygous recessive individuals have a profound deficiency (usually greater than 90% reduction) in the activity of the pivotal enzyme in most tissues. Antemortem diagnosis can be made by enzyme assay of whole skin, cultured skin fibroblasts, purified leukocytes, and in some species, serum [ 10,281. Postmortem diagnosis is made most reliably by enzyme assay of brain (cortex) and liver. Enzyme activity is retained for months in tissues stored at -4" C. Biochemical diagnosis of recessive genotype can be made in utero by amniocentesis and before onset of clinical signs in the neonate. Assay of enzyme activity in tail tips removed aseptically during the first few days of life has been a useful procedure in the management of feline GM1 and GM2 gangliosidosis colonies for research. Tissue of heterozygotes contain about 50%of normal enzyme activity but individual variation requires that prediction of heterozygous genotype by enzymology must be based upon highly standardized methods of sample collection and assay. While it is possible to use whole skin homogenate or leukocytes isolated from peripheral blood, experience with the feline gangliosidoses indicates that cultured fibroblasts provide the most reliable sample. Data in table I11 illustrate enzyme values of cultured fibroblasts from cats of dominant, heterozygous and recessive genotypes from feline GM1 and GM2 gangliosidoses colonies. Research Applications Early research on the lysosomal storage diseases was limited to clinicopathological observations of individual human patients. In recent years, biochemical and morphological study of autopsy material has been augmented by use of cultured 644 Baker el a1 fibroblasts, brain biopsies and tissues from therapeutically aborted homozygous recessive fetuses. While this approach has provided valuable insight, it suffers from serious restrictions on the application of complex research procedures to human subjects. The recent initiative by the National Institutes of Health emphasizing research on rare human genetic diseases intensifies the need to develop model systems that circumvent the limitations imposed on the use of human patients. Investigators in this field have appealed consistently for development and use of cell culture systems and animal models. Useful models must be well defined, easily manipulated, relevant to analogous human disease processes, and readiIy available. Special advantage is gained from models that provide parallel in vitro and in vivo systems. In some instances it has been possible to simulate the metabolic or pathological consequences, or both, of inherited diseases by perturbing normal cultured cells or animals [23, 371. Induction of simulated lysosomal disease has been accomplished by lysosomal loading with the substrate of interest or chemical block of the pivotal enzyme system. While such systems can be useful, they rarely approach the value of spontaneous animal disease analogs. Regretably, relatively few animal analogs of human lysosomal diseases have been identified and thoroughly characterized. About I 1 diseases affecting one or more species have been reported that are thought to be analogous to lysosomal storage diseases of man . All of these diseases are associated with degenerative disorders of the nervous system and nine have been noted in domestic cats. The complete metabolic defects operating in these animal analogs have been elucidated only for the gangliosidoses, canine globoid cell leukodystrophy, bovine mannosidosis and feline mucopolysaccharidosis. Animal analogs of the gangliosidoses fulfill many of the requirements for useful research model systems. Cats with GM1 and GM2 gangliosidoses, swine with GM2 gangliosidosis and dogs with GM1 gangliosidosis are being maintained in laboratory colonies for use in biomedical research. Those herds of cattle that have produced calves with GM gangliosidosis apparently continue to be maintained in Ireland; presumably it may be possible to procure heterozygous breeding stock from these sources. Breeding stock of German Shorthaired Pointers with canine GM2 gangliosidosis, however, has not been maintained for research use. Use of farm animals in research presents substantial difficulties in maintenance of such species, particularly in breeding colonies. This is an especially important limitation in the bovine disease because of the low fecundity and large body size of this species. Swine present fewer problems because of their high reproductive capacity and the opportunity to transfer the mutant GM2 gangliosidosis gene to miniature breeds. There is much to recommend feline gangliosidoses as models for research: 1) availability of established research colonies; 2) extensive characterization of the feline diseases; 3) remarkably close and specific analogy with diseases in children; 4) high reproductive capacity of cats; 5 ) ease of laboratory maintenance and handling of cats; 6) body size which facilitates clinical observations, surgical manipulations, testing and treatment procedures, and availability of reasonable volumes of tissues Gangliosidoses 645 and body fluids; 7) unrivaled position of cats as the favorite species for neurological research and the vast repository of data on the feline nervous system; and 8) availability of well characterized companion cell culture systems. Animal models of the gangliosidoses are particularly valuable for research aimed at defining the pathogenesis of neuronal dysfunction caused by lysosomal disease and for evaluating promising therapeutic methods. Despite advances in understanding the biochemical lesions of lysosomal storage diseases, surprisingly little is known about the relationships between disrupted catabolism of complex metabolites, lysosomal hypertrophy and cell injury or death. Because neurological disease is such a prominent and important part of most lysosomal diseases it is important to understand the specific effect of lysosomal hypertrophy on neuronal dysfunction and death. Demyelination secondary to functional disturbances of Schwann cells has been proposed as a primary factor [ 181, but the complex neurological manifestations and lack of marked demyelination until late in the disease process makes this explanation insufficient. The gangliosidoses are known to be associated with changes in the shape and size of certain neurons. In studying cortical biopsies from children with Tay-Sachs disease (infantile GM2 gangliosidosis), juvenile GM2 gangliosidosis and Hurler’s disease (mucopolysaccharidosis,type I), large neuronal processes (meganeurites) were found between the perikaryon and axon of cortical pyramidal neurons (321. In some neurons the volume of meganeurites exceeded that of the associated soma. Recently, meganeurites comparable to those seen in children have been seen in many areas of brain from cats with GM, gangliosidosis (fig. 8) [30, 3 11. The discovery of abnormal neuronal morphology in gangliosidoses forms the basis for advancing a hypothesis to explain neuronal dysfunction caused by lysosomal hypertrophy . This hypothesis is based on the generally accepted view that the geometric features of multipolar neurons are important in determining the integrative electrophysiological effects of spatially distributed synaptic inputs. Thus the output of neurons conceivably can be altered by abnormal cell morphology such as generalized increases in cell size, or by regional expansions such as meganeurites. Furthermore, if aberrant synaptic inputs associated with meganeurites are functional, the integrative function of affected neurons could be altered profoundly. The formation of neurites in mature neurons engorged with ganglioside also suggests the role of these compounds in neurite induction during normal neuronal differentiation. Continued study of the pathogenesis of the animal gangliosidoses thus provides an unprecedented opportunity to advance understanding of several important aspects of neuronal function in health and disease. In the past, treatment of individuals with most inherited metabolic diseases has been palliative only. In search of alternative methods for treating inborn errors of metabolism, dietary control of substrates and substrate precursors has been suggested as a method to prevent the pathological accumulation of toxic substrate. Dietary therapy has been successful in preventing clinical disease in at least 20 other disorders of amino acid metabolism . While this approach has been extremely effective in 646 Baker era/ ' 25rm ' Fig. 8 Camera lucida drawings of entorhinal cortex pyramidal cells from cat with GMI gangliosidosis. Soma (S)and meganeurite (M). From Purpura and Baker, Brain Research 143 13-26, 1977, with permission of Elsevier, North Holland Biomedical Press. those disorders where restriction of the offending metabolite is feasible, most lysosoma1 storage diseases do not lend themselves to dietary treatment. Diseases such as the gangliosidoses involve faulty degradation of complex molecules which are necessary for life and cannot be controlled by dietary restriction. The thrust of current research is directed toward the development of corrective therapy for these enzymatic deficiencies. An ideal cure for inherited disorders would be substitution of normal DNA coding for synthesis of the defective gene product. Current concepts and the potential for gene therapy recently have been reviewed [ 15). While promising therapeutically, progress in gene therapy will be slow because of technological and ethical restrictions. The theoretical possibility of correcting lysosomal diseases by enzyme replacement was recognized soon after the pathogenetic basis for these diseases was first advanced . The rationale for this approach is based on the assumption that endocytosed replacement enzymes would be brought into direct contact with diseased lysosomes and function in degradation of accumulated substrate. Attempts at in vivo enzyme replacement for lysosomal storage disease have been reviewed [8, 361. Although the progress in enzyme replacement therapy has been significant, the optimistic goal of effective therapy for human lysosomal storage disease has not been realized. Major Gangliosidoses 647 obstacles that must be overcome if enzyme replacement is to be effective include: 1) availability of stable enzymes with high specific activities for natural substrates; 2) protection of replacement enzyme from bioinactivation and immunological reactivity; 3) perfection of methods to deliver replacement enzyme to target pathologic sites; and 4) development of well defined, representative mammalian model systems to test therapeutic methods. In vitro and in vivo systems of the feline gangliosidoses are being used effectively in addressing fundamental aspects of enzyme replacement therapy, such as biological carriers of replacement enzyme; organ, cell and intracellular targeting; enzyme-cell interactions; reversibility of lysosomal hypertrophy; and penetration of endothelial barriers. Lysosomal hypertrophy and retarded catabolism of glycoproteins have been seen in fibroblasts cultured from cats with feline gangliosidoses. Incorporation of ( 14C)galactose into the glycopeptides stored in lysosomes of feline GM1 gangliosidosis provides a predictable and sensitive system to evaluate quantitatively the effects of enzyme replacement therapy. With this system it has been demonstrated that exposure of mutant fibroblasts to liposomes carrying hydrolytically active /I-galactosidase resulted in clearance of more than 80% ''C-glycopeptide and an increase of /3-galactosidase activity to 70% normal within 240 hours 1351. Research on therapy for lysosomal storage diseases is at the threshold of development and many questions remain. It is clear, however, that the animal gangliosidoses will provide the necessary models for exploration of this fundamental and exciting research. 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