Departments of Cell Biology, 2Pathology,and 3Neuroscience, AlbertEinstein College Medicine, New York, NY 10461, USA and 4Lysosomal Diseases Research Unit,The Womens and Childrens Hospital, Adelaide,South Australia, 5006, Australia
Received on April 26, 1999. revisedon June 15, 1999; accepted on June 15, 1999.
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Abstract |
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Introduction |
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The mucopolysaccharide (MPS) storage diseases represent onebroad category of lysosomal disorder in which enzymes needed todegrade glycosaminoglycans are deficient. Proteolytic cleavage ofcellular proteoglycans generates glycosaminoglycans (dermatansulfate, heparan sulfate, keratan sulfate, and chondroitin sulfate)which are normally catabolized by 10 different lysosomal enzymes(33Neufeld and Muenzer, 1995).Numerous types of MPS disease are recognized on the basis of specificenzyme deficiencies and storage of one or more glycosaminoglycans.Sanfilippo syndrome or mucopolysaccharidosis type III is the mostcommon form of MPS. Estimates of incidence range from 1:24,000 inThe Netherlands (van de Kamp, 1981), to 1:66,000 in Australia (28
Meikle et al., 1999) toapproximately 1:324,000 in British Columbia (27
Lowry et al., 1990). There are four subtypesof MPS III that result from deficiencies in different enzymes requiredto degrade heparan sulfate in the lysosome: glucosamine-N-sulfamidasein MPS III A,
-N-acetylglucosaminidasein MPS III B, acetyl-CoA acetyltransferase in MPS III C, and N-acetylglucosamine-6-sulfatasein MPS III D. MPS III A is the most common subtype in Northern Europe,whereas MPS III B is more prevalent in Italy and Greece (Betris,1986; 30
Michelakakis et al.,1995). The genes coding for MPS III A, III B, and IIID have been cloned (35
Robertson et al., 1992; 36
Scott et al., 1995; 22
Karageorgos et al., 1996; 48
Weber et al., 1996; 50
Zhao et al., 1996), and mutations causing MPSIII A in humans have been described previously (4
Blanch et al., 1997; 5
Bunge et al., 1997; 49
Weber et al., 1997; 10
DiNatale et al., 1998).
All subtypes of MPS III result from defective degradation andsubsequent storage of heparan sulfate in the lysosome (33Neufeld and Muenzer, 1995). After a shortperiod of normal development, affected individuals exhibit a rangeof symptoms that may include loss of social skills with aggressivebehavior and hyperactivity, mental retardation, disturbed sleep,coarse facies, hirsutism, and diarrhea. In profoundly affected children, hearingloss and delayed speech development are often present at 2 yearsof age. Skeletal pathology, typical for other types of MPS disease,is relatively mild and often develops after the clinical diagnosisis established. However, there has been considerable variation reportedin the age of onset and the severity of clinical phenotypes observedfor MPS III patients.
There have been four animal models described for MPS III. ANubian goat model for MPS III D (21Jones et al., 1998) has provided valuable clinical,biochemical and morphological detail to assist comparison with humanMPS III D; MPS III B has been described in emu (Giger, 1997) andin a mouse with a targeted mutation (25
Liet al., 1998); MPS III A has been describedin dog (12
Fischer et al.,1998). We report here the discovery of a murine modelof MPS III A that exhibits a profound deficiency of lysosomal sulfamidase(EC 3.10.1.1) activity, and many of the biochemical, pathological,and clinical features found in children with this disease.
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Results |
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Mice with lysosomal storage disease were routinely identifiedby light microscopy of muscle biopsy sections. Fibroblasts of affectedmice had vacuolated and enlarged cytoplasm. When complete litterswere biopsied, affected mice represented about 25% of progeny,indicating autosomal recessive inheritance. Consistent with thiswas the fact that some biopsy negative mice (presumed wild-type)did not produce any biopsy positive progeny in a complete litterwhen mated to a biopsy positive mouse, whereas others (presumedheterozygotes) produced about 50% affected progenyfrom a biopsy positive mating.
At birth, affected pups were indistinguishable from littermates.No significant differences in growth rate or appearance were observeduntil 67 months when affected mice were noted to be lessactive. At this time, the coats of affected animals appeared scruffyand the mice had a hunched posture and abdominal distension (Figure 1A). Males or females caged together (up to5 per cage) did not show any overtly aggressive behavior. At ~7months of age the clinical onset of corneal opacity was often notedin affected mice. By about 710 months affected mice died.Among 30 male and female mice the average age of death was 7.2 months(range 3 to 10.5 months). A few mice lived from 1214 months.At death, mice invariably exhibited a grossly distended bladderfilled with 12 ml of turbid urine, and they also had hepatosplenomegaly (Figure 1B).
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Lysosomal storage was dramatically evident in the liver (Figure 2C) and to a lesser extent in the spleen (notshown). Kupffer cells of the hepatic sinusoids were swollen withcytoplasmic vacuoles and randomly clustered together. In advancedcases the hepatocytes tended to develop microvesiculated cytoplasm.The splenic parenchyma contained microvesiculated cellsin the dense connective tissue trabeculae and sinuses and in perivascularlocations.
Terminally the mice developed urinary bladder distension (Figure 1B), often accompanied by unilateral or bilateral hydronephrosis.The kidney was altered by accumulation of storage material primarilyin the cortical regions (Figure 2D). Epithelialpodocytes of the glomerular tuft exhibited microvesiculated cytoplasm.Cytoplasm of distal convoluted tubules was microvesiculated, whereasproximal convoluted tubules appeared unaffected, or at most onlymildly so. Interstitial cells were diffusely affected, characterizedby microvesiculated cytoplasm. Epithelial cells lining collectingtubules had microvesiculated cytoplasm in the thick ascending limb,collecting ducts, thin descending limb and medullary thick ascendinglimb. The wall of the urinary bladder was thickened grossly, andmicroscopically the submucosa was distorted and expanded by infiltrationof fibroblasts and macrophages with abundant cytoplasm containingPAS positive material (Figure 2E).
Cardiac muscle was often markedly affected in chronic cases.Microvesiculated fibroblasts and macrophages commonly expanded themyocardial endomysium and perivascular spaces (Figure 2F). In advanced cases myocardiocytes underwentdegenerative changes and were replaced by fibroblasts with foamycytoplasm. Microvesiculated cells also infiltrated the subendothelialconnective tissue core of valvular cusps and arterial perivascularspaces.
Bone deformation is commonly reported in the mucopolysaccharidoses(33Neufeld and Muenzer, 1995).The calvarium was abnormally thickened in all affected mice whencompared to controls. Vertebral deformation was often the most severe lesion,and frequently cartilagenous matrix of particularly the thoracicvertebrae, proliferated within the spinal canal. Chondrocytes hadmicrovesiculated cytoplasm, as did some periosteal cells.
Urine analysis identifies accumulation of heparansulfate in affected mice
Urine glycosaminoglycans (GAGs) from affected and control micewere analyzed by high-resolution electrophoresis. Whereas controlmouse urine had a mixture of mostly chondroitin sulfate, with heparansulfate and dermatan sulfate, affected mice had predominantly heparansulfate (Figure 4). The pattern in affectedmice was typical of patients that have Sanfilippo syndrome (18Hopwood and Harrison, 1982). The gradientgel electrophoresis pattern obtained for urine and brain GAGs fromaffected mice was also typical of patients with MPS III A (see belowand Figure 5).
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The data in Table show significantelevation of ß-hexosaminidaseactivity in liver and ß-glucuronidaseactivity in brain. Specific activities of enzymes responsible forGAG degradation were also increased about 2-fold, with the notable exceptionof sulfamidase. Sulfamidase activity was markedly deficient (34% ofthe specific activity in control mice). Therefore, sulfamidase wasthe only hydrolase among those that give rise to heparan sulfateaccumulation and Sanfilippo syndrome that was severely reduced inactivity in affected mice.
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Digestion of isolated GAGs with recombinant human sulfamidasebefore gradient gel electrophoresis (6Byers et al., 1998), provided a definitive diagnosisof a deficiency of sulfamidase as the cause of heparan sulfate storageand the clinical phenotype in the mouse reported here. A shift inbanding pattern to more rapidly migrating species was observed aftersulfamidase digestion, reflecting a change in charged species dueto the replacement of negatively charged, nonreducing end, glucosamine-N-sulfate residues by a positively charged glucosamineresidue (Figure 5).
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Discussion |
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Two cases of MPS III A have been reported in dogs (12Fischer et al., 1998).These animals exhibited pelvic limb ataxia as young adults whichprogressed over several years to severe cerebellar ataxia. Mildcerebral and cerebellar atrophy was found and neurons in many brainregions exhibited substantial intracellular storage. Purkinje cellsof the cerebellum were particularly affected and widespread lossof these cells apparently contributed to the clinically-evidentataxia. In the MPS III A mice, there is less obvious motor systemdysfunction and Purkinje cells are not lost in substantial numbers.Like the dog model, there is widespread neuronal storage, with the ultrastructureof the storage material being similar to that reported in the dog.In viscera of the dog model, fibroblasts, hepatocytes, and renaltubular cells were vacuolated. For the mice, only distal renal tubuleswere severely affected, rather than both proximal and distal asin the dog. However, in both mouse and dog the urinary bladder wallwas conspicuously thickened. Changes in cardiac muscle observedin the MPS III A mice (Figure 2F) were notdescribed in the dog model.
The MPS III A mouse should be useful for investigations of thecell biological and neurological consequences of all mucopolysaccharidoses,including MPS III A. A wide variety of functions have been suggestedfor different GAGs and for individual proteoglycans, particularlyin the developing and aging brain (37Small et al., 1996). When added to culturedcortical neurons, for example, heparan sulfate induced the formationof long singular axons but few or no dendrites (7
Calvet et al., 1998). In contrast, the additionof dermatan sulfate increased dendrite growth, possibly throughchanges in adhesion properties of the growing neurites. Heparansulfate also constitutes the major GAG sidechain of proteoglycanslike syndecan, glypican, and cerebroglycan, that have been implicatedin a variety of functions, including modulation of growth factorreceptorinteractions (37
Small et al.,1996).
In addition to storing GAGs, most forms of MPS disease are knownto store gangliosides. Thus, not only does heparan sulfate accumulatein brain tissue of humans and dogs affected by MPS III A, but abnormalamounts of GM2 and GM3 gangliosides also occur (8Constantopoulos et al., 1980; 20
Jones et al., 1997; 12
Fischer et al., 1998). The MPS III A mice reportedhere are similar in that abnormal accumulation of GM2 ganglioside wasdetected in neurons of the cerebral cortex. It has been proposedthat ganglioside storage in MPS disease may be due to secondaryinhibition of ganglioside specific neuraminidases by accumulatedsulfated GAGs (1
Baumkotter and Cantz, 1983; 20
Jones et al., 1997, 1998).The degree of ganglioside accumulation in these diseases often mimicsthat of the primary ganglioside storage disorders and the overabundanceof particular gangliosides may be responsible for some aspects ofbrain dysfunction, including mental retardation (42
Walkley,1995, 1998; 46
Walkley et al., 1995). Availability of the MPS III A modelin mice will be useful for elucidating the relationship betweenthe primary enzyme deficiency, secondarily-induced biochemical abnormalities,and neuronal dysfunction leading to clinical neurological disease.
Animal models of storage diseases are also useful for testing treatmentstrategies. In several animal models of lysosomal disorders thedevelopment of clinical disease can be ameliorated by both enzymereplacement therapy and bone marrow transplantation (see 45Walkley et al., 1994; 9
Crawley et al., 1996; 43
Walkley, 1998). In theory, many more lysosomalstorage diseases could be treated by such therapies since lysosomal hydrolaseswith mannose-6-phosphate residues amongst their N-linkedglycans, bind to cell surface mannose-6-phosphate receptors andare delivered to lysosomes following receptor-mediated endocytosis(31
Neufeld, 1980; 23
Kornfeld,1986). Similarly, lysosomal enzymes bearing terminal mannoseor galactose residues can be efficiently endocytosed by certaincell types (34
Rattazzi and Dobrenis, 1991).The existence of these pathways to the lysosome provides the rationalefor lysosomal hydrolase enzyme replacement therapy. Provided a lysosomal hydrolaseis processed with the correct N-linked glycans,it can be targeted to lysosomes following injection into the bloodstream.Effective uptake and delivery to lysosomes can also be achievedthrough modifications such as the use of the C fragment of tetanustoxin to specifically enhance targeting to neurons (11
Dobrenis et al., 1992). Because of the bloodbrain barrier,lysosomal enzymes are unlikely to enter the brain from the circulation.Therefore, alternate strategies must be developed to target lysosomalenzymes to sites of pathology in the brain. One potential meansof delivery to brain is via bone marrow transplantation. In thiscase, donor bone marrow-derived monocytes are believed to enterthe brain where they differentiate as microglia and serve as a potentialsource of missing lysosomal hydrolases (45
Walkleyet al., 1994; 24
Krivit et al., 1995; 47
Walkley et al., 1996). Differences in the secretionand/or stability of secreted enzymes by such cells is alikely explanation for the variable success of this technique inthe treatment of a variety of storage diseases (44
Walkleyand Dobrenis, 1995). Over the years numerous models oflysosomal hydrolase deficiency have been identified from spontaneousmutations in animal populations and, more recently, additional modelshave been generated by targeted gene mutation in the mouse (reviewedin Jolly and Walkley, 1997; 38
Suzuki andProia, 1998). The murine model of MPS III A describedhere will be a valuable tool to determine effective treatment strategiesfor this and related storage diseases.
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Materials and methods |
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Histology, electron microscopy (EM), and immunostaining
For morphological studies using light microscopy animals weredeeply anesthetized with pentobarbital and perfused via an intracardiaccatheter with 4% paraformaldehyde in 0.1 M phosphate buffer,pH 7.2. For EM analysis, tissues were postfixed in 4% paraformaldehydeand 2% glutaraldehyde in 0.1M phosphate buffer, pH 7.2.Paraffin and Epon embedding, for light and electron microscopicstudies respectively, were carried out using routine methods. Forimmunocytochemical studies, 40 µm sectionswere cut on a vibratome. Monoclonal antibodies to LAMP1 (40Uthayakumar and Granger, 1995) from theDevelopmental Studies Hybridoma Bank, The University of Iowa, GM2ganglioside (a gift from Dr. Philip Livingston, Memorial Sloan Kettering),and F4/80 (Serotec) were applied at predetermined dilutionsfollowed by indirect immunolabeling using appropriate bridging antibodies.Peroxidaseantiperoxidase labeling was detected with diaminobenzidene (Sigma,St. Louis, MO) using routine histochemical procedures (42
Walkley, 1995).
Lysosomal enzyme assays
Tissue homogenates were prepared by two methods. In method 1,tissues were homogenized on ice in 1020 volumes 0.1 Mcitrate buffer, pH 5.5, with 25 strokes of a glass-to-glass tissue homogenizer(Kontes #88550000022) mounted on an EberbachCon-Torque tissue grinder. Homogenates were centrifuged for 15 minat 10,000 x g and supernatantsrecovered for enzyme assay and protein determination. Tissuehomogenates were assayed in triplicate with fluorogenic 4-methylumbelliferyl (4-MU) substrates at 37°Cbased on standard protocols (Galjaard,1980). ß-N-Acetyl-D-hexosaminideN-acetylhexosamino-hydrolase (ß-hexosaminidase)(E.C. 3.2.1.52) was assayed at pH 4.5 for 0.5 h with 5 mM 4-MU-2-acetamidodeoxyD-glucopyranoside in 0.2 M citric acid0.34M dibasic sodium phosphate;
-D-mannosidemannohydrolase (
-mannosidase) (E.C.3.2.1.24) was assayed at pH 3.75 for 1 h with 4 mM 4-MUD-mannopyranoside in 0.1 M citric acid-0.2 M dibasicsodium phosphate buffer with 1.5 mM ZnCl2; GM1 ganglioside ß-galactosidase (ß-galactosidase)(E.C. 3.2.1.23) was assayed at pH 4.4 for 1 h with 1 mM 4-MU-D-galactopyranosidein 0.1 M citric acid0.2 M dibasic sodium phosphate bufferwith 100 mM NaCl; and ß-D-glucuronideglucuronohydrolase (ß-glucuronidase)(E.C. 3.2.1.31) was assayed at pH 4.8 for 1 h with 10 mM 4-MUD-glucuronide in 0.1 M acetate buffer. Resultsare expressed as nanomoles substrate cleaved per hour per mg proteinassayed by the Lowry method (26
Lowry et al., 1951).
In method 2, tissues were homogenized in 0.1% TritonX-100 (v/v), freeze-thawed 3 times and sonicated for 10sec three times (Ystrom Systems USA, power setting 7), centrifuged5 min at 500 x g andthe supernatant assayed on the same day. Assays were performed essentiallyas described previously (16Hopwood and Elliott,1982) in 50 mM sodium acetate buffer, pH 5, with 34 µM tetrasaccharide substrate (glucosamine-N-sulfate-(1,4)-iduronicor glucuronic acid-(1,4)-glucosamine-N-sulfate-(1,4)-{13H} idonic,gluconic, or anhydroidonic acid) in a final volumeof 12 µl. Each assay contained 30 µg protein measured by the Lowry method(26
Lowry et al., 1951) comparedto Dade Human Protein Standard (Baxter Healthcare Corp., USA) andwas incubated at 37°C for 16 h.
-N-Acetylglucosaminidase was measuredas described (16
Hopwood and Elliott, 1982)using 60 µM disaccharide substrate (N-acetylglucosaminide-(1,4)-{13H}-idonic,-gluconic, or -anhydroidonic acid) in 50 mM sodium acetate pH 4.5with 2030 µg protein for 7h at 37°C. Acetyl-CoA glucosamine N-acetyltransferaseand glucosamine-6-sulfatase were assayed using a monosaccharideand a disaccharide substrate, respectively, as previously published(17
Hopwood and Elliott, 1981; 13
Freeman and Hopwood, 1989). Iduronate-2-sulfataseactivity was assayed using a disaccharide substrate (15
Hopwood,1979).
High-resolution and gradient gel electrophoresisof glycosaminoglycans isolated and treated with sulfamidase
Glycosaminoglycans (GAGs) were isolated from urine and tissuesas described previously (18Hopwood and Harrison,1982; 6
Byers et al.,1998). The amount and types of GAGs present in urine wereassayed using high resolution electrophoresis (18
Hopwoodand Harrison, 1982) or gradient gel electrophoresis (39
Turnbull et al., 1997; 6
Byers et al., 1998). GAGfractions were either subjected (or not) to digestion with recombinant humansulfamidase and run on 3040% linear gradientpolyacrylamide gels as described (6
Byers et al., 1998). GAG samples (1025 µl) containing 0.55.7 µg of uronic acid, and marker standards(510 µl) were combined with10% glycerol and trace amounts of phenol red and bromophenolblue. Electrophoresis was performed at 350V for 16 h or until thephenol red dye front was 1 cm from the bottom of the gel. Oligosaccharideswithin the resolving gel were stained with alcian blue/silver(29
Merril et al., 1981).
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Acknowledgments |
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Abbreviations |
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Note Added in Proof |
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Footnotes |
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References |
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