A mouse model for mucopolysaccharidosistype III A (Sanfilippo syndrome)

Mantu Bhaumik, Vivienne J.Muller3, Tina Rozaklis3, Linda Johnson2, Kostantin Dobrenis3, Riddhi Bhattacharyya, Sarah Wurzelmann3, Peter Finamore3, John J.Hopwood4, Steven U.Walkleya,3 and Pamela Stanleya

Departments of Cell Biology, 2Pathology,and 3Neuroscience, AlbertEinstein College Medicine, New York, NY 10461, USA and 4Lysosomal Diseases Research Unit,The Women’s and Children’s Hospital, Adelaide,South Australia, 5006, Australia

Received on April 26, 1999. revisedon June 15, 1999; accepted on June 15, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Note Added in Proof
 References
 
Mucopolysaccharidosis type III A (MPS III A, Sanfilippo syndrome)is a rare, autosomal recessive, lysosomal storage disease characterizedby accumulation of heparan sulfate secondary to defective functionof the lysosomal enzyme heparan N-sulfatase (sulfamidase).Here we describe a spontaneous mouse mutant that replicates manyof the features found in MPS III A in children. Brain sections revealedneurons with distended lysosomes filled with membranous and floccularmaterials with some having a classical zebra body morphology. Storagematerials were also present in lysosomes of cells of many othertissues, and these often stained positively with periodic-acid Schiffreagent. Affected mice usually died at 7–10 months of age exhibitinga distended bladder and hepatosplenomegaly. Heparan sulfate isolatedfrom urine and brain had nonreducing end glucosamine-N-sulfateresidues that were digested with recombinant human sulfamidase.Enzyme assays of liver and brain extracts revealed a dramatic reductionin sulfamidase activity. Other lysosomal hydrolases that degradeheparan sulfate or other glycans and glycosaminoglycans were eithernormal, or were somewhat increased in specific activity. The MPSIII A mouse provides an excellent model for evaluating pathogenicmechanisms of disease and for testing treatment strategies, includingenzyme or cell replacement and gene therapy.


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Note Added in Proof
 References
 
Lysosomal storage diseases are rare, autosomal recessive diseasesthat arise from a reduction in activity of one or more of the lysosomalhydrolases responsible for the catabolism of a wide variety of lipids,glycans, or proteins (32GoNeufeld, 1991). Theresultant disruption in specific catabolic pathways leads to theaccumulation of undegraded materials within lysosomes which causeslysosomal engorgement and cell swelling. The medical consequencescan be devastating as many lysosomal storage diseases lead to severeneurological impairment and to major organ dysfunction. Death iscommon at an early age.

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(33GoNeufeld 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 (28GoMeikle et al., 1999) toapproximately 1:324,000 in British Columbia (27GoLowry 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, {alpha}-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; 30GoMichelakakis et al.,1995). The genes coding for MPS III A, III B, and IIID have been cloned (35GoRobertson et al., 1992; 36GoScott et al., 1995; 22GoKarageorgos et al., 1996; 48GoWeber et al., 1996; 50GoZhao et al., 1996), and mutations causing MPSIII A in humans have been described previously (4GoBlanch et al., 1997; 5GoBunge et al., 1997; 49GoWeber et al., 1997; 10GoDiNatale et al., 1998).

All subtypes of MPS III result from defective degradation andsubsequent storage of heparan sulfate in the lysosome (33GoNeufeld 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 (21GoJones 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 (25GoLiet al., 1998); MPS III A has been describedin dog (12GoFischer 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Note Added in Proof
 References
 
Characteristics of affected mice
During the course of breeding mice generated from an embryonicstem cell clone WW6.186 that transmitted a targeted mutation inthe Mgat3 gene to CD1 mice (3GoBhaumik et al., 1998), a 14 month male homozygousfor the Mgat3-/- mutation was observed to bewalking in circles and to be scruffy and ill in appearance. Sectionsprepared from the brain of this mouse showed dramatic alterationsin lysosomal morphology and numerous zebra bodies characteristicof lysosomal storage disease. When additional Mgat3-/- micewere examined, only about a third were found to be affected. Subsequently,the same brain lesions were discovered in two Mgat3+/+ mice.From the latter wild type mice, a colony that has produced ~150affected mice was established. These mice are of mixed genetic backgroundincluding predominantly 129SvJ and CD1 with some C57Bl/6and SJL strain contributions.

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 6–7 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 7–10 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 12–14 months.At death, mice invariably exhibited a grossly distended bladderfilled with 1–2 ml of turbid urine, and they also had hepatosplenomegaly (Figure 1B).



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Fig. 1. (a) Photographof an adult mouse (age 9 months) affected by MPS III A (left) anda normal littermate (right). (b) Affected adultmouse showing distended bladder and enlarged liver and spleen.

 
Microscopic evidence of lysosomal storage
Light microscopic analysis of brain and other tissues revealed widespreadand variable intracellular storage in a variety of cell types (Figure 2). The overall degree of intracellular storage inbrain varied with age, with the oldest animals exhibiting the greatestextent of accumulated material. Neurons within the cerebral andcerebellar cortices, the deep cerebellar nuclei, and other brainareas exhibited cytoplasmic distension with vacuoles containingmaterial that often stained positively with PAS (Figure 2A). Toluidine blue staining of 2 µmplastic sections taken from the cerebral cortex revealed differenttypes and degrees of storage in different brain cells (Figure 2B). With light microscopy, cortical neuronscharacteristically exhibited dense inclusions which stained positivelywith toluidine blue, whereas adjacent glial cells typically exhibiteda vesiculated appearance. Immunocytochemical staining of brain tissue usingantibodies to LAMP1 revealed that storage predominated within thelysosomal system. In addition, storage material in many types ofcells also frequently stained with antibodies to GM2 ganglioside(data not shown).



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Fig. 2. Light microscopic analysisof storage in cells of various tissues. (A) PASstain of a paraffin section showing neurons in a deep cerebellarnucleus. Arrows indicate PAS-positive material within neurons (125x). (B) Toluidine bluestained 2 µm thick plastic section ofcerebral cortex. Upper right and lower arrows indicate neurons withcytoplasmic storage; upper left arrow illustrates the presence ofa typical perineuronal glial cell with vesiculated cytoplasm. (80x). (C) Toluidine bluestained 2 µm thick plastic section ofliver. Kupffer cells demonstrate the greatest degree of storage.Arrows show vesiculation (80x). (D)Toluidine blue stained 2 µm thick plasticsection of kidney showing storage in tubules. Distal tubules (upperarrows) showed substantially more storage than proximal tubules (lowerarrows) (80x). (E)PAS stain of paraffin section showing storage within cells infiltratingthe submucosa of the urinary bladder (arrows) (80x)(F) H&E stain of paraffin section of cardiacmuscle illustrating massive infiltration of cells with foamy cytoplasmand disruption of normal tissue architecture (arrows). (80x).

 
Electron microscopic (EM) analysis was necessary to identify thenature of the inclusions. EM of cerebral cortex revealed that neuronscontained typical "zebra body" type storage materialor inclusions with a more floccular characteristic. Most characteristically,combinations of these inclusions were found mixed within individualneurons (Figure 3A). Other cells in thebrain parenchyma resembling microglia and perineuronal satellite cellscontained clear, electron-lucent inclusions (Figure 3B).



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Fig. 3. Electron microscopy of cellsin cerebral cortex. (A) Electron micrograph oftypical storage within a cortical neuron like those shown in Figure2B. Storage vacuoles ranged from those containing floccular materialto others with stacks of membrane (arrowhead) sometimes resemblingzebra bodies. (7920x). (B) Electronmicrograph of a perineuronal glial cell showing characteristic vesiculatedcytoplasm (arrowhead). (4680x). G, Golgiapparatus; N, nucleus.

 
Mesenchymal cells of the leptomeninges, perivascular spacesand endothelium were remarkable for exhibiting vesiculated cytoplasmwhich also gave positive staining with PAS. Many of these same cells,particularly those in the pial areas, also stained with antibodiesto F4/80 indicating a monocytic lineage (data not shown).Microvesiculated cytoplasmic distension of endoneural fibroblasts,endothelial cells and scattered Schwann cells were prominent indorsal root ganglia and peripheral nerves. Neurons of the dorsalroot ganglia, like those of the CNS, exhibited fibrillogranularand zebra body type inclusions. Neither axonal or myelin alterationswere observed in peripheral nerves. In the eye, rare corneal substantiapropria fibroblasts had microvesiculated cytoplasm. Retinal pigmentepithelial cells, ciliary epithelium and scleral fibroblasts oftenwere identified with vacuolated cytoplasmic material. Cells withmicrovesiculated cytoplasmic distension randomly infiltrated theear, affecting the osseous labyrinth and fibrovascular stalk ofthe tympanic membrane.

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(33GoNeufeld 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 (18GoHopwood 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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Fig. 4. High resolution electrophoresisof urine GAGs. Samples were prepared and analyzed as described in Materials and methods. Control contains GAGs from anequal volume mixture of urine from a patient with MPS II (deficientin iduronate-2-sulfatase) and a patient with MPS-IVA (deficientin N-acetylgalactosamine-6-sulfatase); Affected lanes contain GAGsfrom urine of an affected mouse at 1x and0.5x dilution, respectively; Normalcontains GAGs from a control, unaffected mouse at 1x.The mice were ~10 months of age.

 


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Fig. 5. Gradient gel electrophoresisof urine and brain GAGs. Samples were prepared and analyzed as describedin Materials and methods from an unaffected humancontrol, a MPS III A patient and an affected MPS III A mouse, age~10 months. Standards were heparan sulfate octa-, hexa-, and tetra-oligosaccharides. Sulfamidase + or – denotestreatment (or not) with recombinant human sulfamidase prior to gelelectrophoresis.

 
Lysosomal hydrolase activities in liver, brain,and kidney
The urinalysis data pointed strongly to Sanfilippo Syndrome as thebasis of lysosomal storage in the affected mice. This syndrome canbe caused by a deficiency in any one of four enzymes: glucosamine-N-sulfamidase (MPS III A), {alpha}-N-acetylglucosaminidase (MPS III B), acetyl-CoAacetyltransferase (MPS III C), or N-acetylglucosamine-6-sulfatase(MPS III D). Therefore, each of these enzymes, as well as iduronic acid-2-sulfatase,another sulfatase that is required for both heparan sulfate anddermatan sulfate degradation, was assayed in various tissue extracts.In addition, several other lysosomal enzymes were analyzed.

The data in Table Go 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 (3–4% 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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Table I. Activity of lysosomal hydrolases in tissues homogenates
 
Nature of nonreducing-end of glycosaminoglycans(GAGs) isolated from mouse MPS III A urine and brain
GAGs were isolated from urine and brain of an affected mouse andcompared to similar preparations from a MPS III A patient and anunaffected human control. Complex banding patterns that showed differencesbetween samples from normal and affected subjects were observedon gradient gel electrophoresis (Figure 5).A comparison particularly in the low molecular weight oligosaccharideregion of the gel, clearly demonstrates the presence of similarGAG patterns between patient and mouse MPS III A samples. We havepreviously reported that the pattern of GAGs obtained using thismethod is different for each MPS type and therefore diagnostic (6GoByers et al., 1998).

Digestion of isolated GAGs with recombinant human sulfamidasebefore gradient gel electrophoresis (6GoByers 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).


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Note Added in Proof
 References
 
In this paper we describe a new mouse model for Sanfilippo disease.The spontaneous mouse mutant we have identified is specificallydeficient in sulfamidase activity, and has heparan sulfate-uria,making this a model for MPS III A. Like patients with MPS III A,the mice have little, if any, sulfamidase activity (Table Go) and they accumulate heparan sulfate thathas glucosamine-N-sulfate nonreducing ends (Figure 5), as expected if sulfamidase is inactive.Other lysosomal hydrolases are for the most part increased in activity,a common characteristic of many lysosomal storage diseases (33GoNeufeld and Muenzer, 1995). In humans,MPS III A is caused by a variety of different inherited mutationsthat reduce the activity of glucosamine N-sulfamidase(4GoBlanch et al., 1997; 5GoBunge et al., 1997; 49GoWeber et al., 1997; 10GoDi Natale et al., 1998).The mouse sulfamidase gene sequence has not been reported. Usinga human sulfamidase cDNA probe and 10 µgpoly(A)+ RNA that gave a strong signal with a control 3kb probe, no mouse sulfamidase transcripts were detected by Northernanalysis (R.Bhattacharryya, unpublished observations). However,the fact that residual activity was evident in tissue extracts fromaffected mice (Table Go) suggests thatthe mouse mutation is likely to be a point mutation that reducessulfamidase activity, similar to the mutations observed in humanswith MPS III A.

Two cases of MPS III A have been reported in dogs (12GoFischer 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 (37GoSmall et al., 1996). When added to culturedcortical neurons, for example, heparan sulfate induced the formationof long singular axons but few or no dendrites (7GoCalvet 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 factor–receptorinteractions (37GoSmall 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 (8GoConstantopoulos et al., 1980; 20GoJones et al., 1997; 12GoFischer 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 (1GoBaumkotter and Cantz, 1983; 20GoJones 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 (42GoWalkley,1995, 1998; 46GoWalkley 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 45GoWalkley et al., 1994; 9GoCrawley et al., 1996; 43GoWalkley, 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(31GoNeufeld, 1980; 23GoKornfeld,1986). Similarly, lysosomal enzymes bearing terminal mannoseor galactose residues can be efficiently endocytosed by certaincell types (34GoRattazzi 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 (11GoDobrenis et al., 1992). Because of the blood–brain 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 (45GoWalkleyet al., 1994; 24GoKrivit et al., 1995; 47GoWalkley 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 (44GoWalkleyand 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; 38GoSuzuki 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Note Added in Proof
 References
 
Experimental animals
A colony of mice with the sulfamidase deficiency was generatedas described in Results. Affected mice were identifiedby microscopic examination of biopsies of the quadriceps femoris muscletaken under local anesthesia. Biopsies were immediately placed in4% paraformaldehyde, left overnight at 4°Cand subsequently embedded by routine methods in Epon for 2 µm sections.Affected animals were identified by light microscopy of toluidineblue stained sections on the basis of heavily vesiculated interstitialcells among muscle fibers. All studies using animals had the approvalof the Institutional Care and Use Committee of the Albert EinsteinCollege of Medicine.

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 (40GoUthayakumar 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.Peroxidase–antiperoxidase labeling was detected with diaminobenzidene (Sigma,St. Louis, MO) using routine histochemical procedures (42GoWalkley, 1995).

Lysosomal enzyme assays
Tissue homogenates were prepared by two methods. In method 1,tissues were homogenized on ice in 10–20 volumes 0.1 Mcitrate buffer, pH 5.5, with 25 strokes of a glass-to-glass tissue homogenizer(Kontes #8855000–0022) 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 (GoGaljaard,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-acetamidodeoxy–D-glucopyranoside in 0.2 M citric acid–0.34M dibasic sodium phosphate; {alpha}-D-mannosidemannohydrolase ({alpha}-mannosidase) (E.C.3.2.1.24) was assayed at pH 3.75 for 1 h with 4 mM 4-MU–D-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 acid–0.2 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-MU–D-glucuronide in 0.1 M acetate buffer. Resultsare expressed as nanomoles substrate cleaved per hour per mg proteinassayed by the Lowry method (26GoLowry 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 (16GoHopwood 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)-{1–3H} idonic,gluconic, or anhydroidonic acid) in a final volumeof 12 µl. Each assay contained 30 µg protein measured by the Lowry method(26GoLowry et al., 1951) comparedto Dade Human Protein Standard (Baxter Healthcare Corp., USA) andwas incubated at 37°C for 16 h. {alpha}-N-Acetylglucosaminidase was measuredas described (16GoHopwood and Elliott, 1982)using 60 µM disaccharide substrate (N-acetylglucosaminide-(1,4)-{1–3H}-idonic,-gluconic, or -anhydroidonic acid) in 50 mM sodium acetate pH 4.5with 20–30 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(17GoHopwood and Elliott, 1981; 13GoFreeman and Hopwood, 1989). Iduronate-2-sulfataseactivity was assayed using a disaccharide substrate (15GoHopwood,1979).

High-resolution and gradient gel electrophoresisof glycosaminoglycans isolated and treated with sulfamidase
Glycosaminoglycans (GAGs) were isolated from urine and tissuesas described previously (18GoHopwood and Harrison,1982; 6GoByers et al.,1998). The amount and types of GAGs present in urine wereassayed using high resolution electrophoresis (18GoHopwoodand Harrison, 1982) or gradient gel electrophoresis (39GoTurnbull et al., 1997; 6GoByers et al., 1998). GAGfractions were either subjected (or not) to digestion with recombinant humansulfamidase and run on 30–40% linear gradientpolyacrylamide gels as described (6GoByers et al., 1998). GAG samples (10–25 µl) containing 0.5–5.7 µg of uronic acid, and marker standards(5–10 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(29GoMerril et al., 1981).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Note Added in Proof
 References
 
We thank Melissa Lopez for technical assistance, Larry Herbstfor performing muscle biopsies and Wendy Norton for the high-resolutionelectrophoresis analysis of mouse urine. This work was supportedby National Institutes of Health Grant R37 30645 from the NCI (toP.S.), RO1 32169 (to S.U.W.), a Program Grant from the NationalHealth and Medical Research Council of Australia (to J.J.H.), andthe WCH Research Foundation (to J.J.H.).


    Abbreviations
 
MPS, mucopolysaccharidosis; EM, electron microscopy; 4-MU, 4-methylumbelliferyl;PAS, periodic acid Schiff; GAG, glycosaminoglycan.


    Note Added in Proof
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Note Added in Proof
 References
 
While this manuscript was in press we identified a point mutationin the sulfamidase gene of affected mice and have shown that mutantcDNA is inactive in CHO cell transfectants (R.Bhattacharrya, B.Gliddon,T.Beccari, G.Yogalingam, M.Bhaumik, J.J.Hopwood and P.Stanley, manuscriptin preparation).


    Footnotes
 
a To whom correspondence should be addressed at:Department of Cell Biology, Albert Einstein College Medicine, 1300Morris Park, New York, NY 10461 Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Note Added in Proof
 References
 
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