Adeno-associated virus serotypes 9 and rh10 mediate strong neuronal transduction of the dog brain.

Canine models have many advantages for evaluating therapy of human central nervous system (CNS) diseases. In contrast to nonhuman primate models, naturally occurring canine CNS diseases are common. In contrast to murine models, the dog's lifespan is long, its brain is large and the diseases affecting it commonly have the same molecular, pathological and clinical phenotype as the human diseases. We compared the ability of four intracerebrally injected adeno-associated virus vector (AAV) serotypes to transduce the dog brain with green fluorescent protein as the first step in using these vectors to evaluate both delivery and efficacy in naturally occurring canine homologs of human diseases. Quantitative measures of transduction, maximum diameter and area, identified both AAV2/9 and AAV2/rh10 as significantly more efficient than either AAV2/1 or AAV2/5 at transducing cerebral cortex, caudate nucleus, thalamus and internal capsule. Fluorescence co-labeling with cell-type-specific antibodies demonstrated that AAV2/9 and AAV2/rh10 were capable of primarily transducing neurons, although glial transduction was also identified and found to be more efficient with the AAV2/9 vector. These data are a prerequisite to evaluating the efficacy of recombinant AAV vectors carrying disease-modifying transgenes to treat naturally occurring canine models in preclinical studies of human CNS disease therapy.

Sodium/glucose cotransporter-1, sweet receptor, and disaccharidase expression in the intestine of the domestic dog and cat: two species of different dietary habit.

The domestic cat (Felis catus), a carnivore, naturally eats a very low carbohydrate diet. In contrast, the dog (Canis familiaris), a carno-omnivore, has a varied diet. This study was performed to determine the expression of the intestinal brush border membrane sodium/glucose cotransporter, SGLT1, sweet receptor, T1R2/T1R3, and disaccharidases in these species adapted to contrasting diets. The expression (this includes function) of SGLT1, sucrase, maltase and lactase were determined using purified brush border membrane vesicles and by quantitative immunohistochemistry of fixed tissues. The pattern of expression of subunits of the sweet receptor T1R2 and T1R3 was assessed using fluorescent immunohistochemistry. In proximal, middle, and distal small intestine, SGLT1 function in dogs was 1.9- to 2.3-fold higher than in cats (P = 0.037, P = 0.0011, P = 0.027, respectively), and SGLT1 protein abundance followed an identical pattern. Both cats and dogs express T1R3 in a subset of intestinal epithelial cells, and dogs, but not cats, express T1R2. In proximal and middle regions, there were 3.1- and 1.6-fold higher lactase (P = 0.006 and P = 0.019), 4.4- and 2.9-fold higher sucrase (both P < 0.0001), and 4.6- and 3.1-fold higher maltase activity (P = 0.0026 and P = 0.0005), respectively, in the intestine of dogs compared with cats. Dogs have a potential higher capacity to digest and absorb carbohydrates than cats. Cats may suffer from carbohydrate malabsorption following ingestion of high-carbohydrate meals. However, dogs have a digestive ability to cope with diets containing significant levels of carbohydrate.

Mucopolysaccharidosis in a cat with arylsulfatase B deficiency: a model of Maroteaux-Lamy syndrome.

A Siamese cat that presented clinical signs similar to those seen in humans with mucopolysaccharidoses was studied. The animal excreted increased amounts of polymeric glycosaminoglycans in the urine, consisting almost entirely of dermatan sulfate. Electron microscopy of circulating polymorphonuclear leukocytes revealed the presence of many membrane-bound lamellar inclusion bodies. Sulfate incorporation studies with cultured skin fibroblasts indicated defective glycosaminoglycan degradation. These cells showed a deficiency in arylsulfatase B activity. The disorder appears similar or identical to the Maroteaux-Lamy syndrome described in humans.

Genetic basis of XX male syndrome and XX true hermaphroditism: evidence in the dog.

Serological analysis of white blood cells from the members of a family of American cocker spaniels indicates that a form of abnormal sexual development, in which individuals with a female karyotype have testes or ovotestes, is caused by anomalous transmission of male-determining H-Y genes.

Clinical characterization of cardiovascular abnormalities associated with feline mucopolysaccharidosis I and VI.

OBJECTIVE: The purpose of this study was to define the cardiovascular abnormalities present in young and adult cats affected with the lysosomal storage diseases mucopolysaccharidosis (MPS) I and MPS VI. METHOD: Eighteen cats affected with MPS I and 10 cats affected with MPS VI were evaluated by physical examination, electrocardiography and echocardiography. Electrocardiography (ECG) was performed on all MPS I and 9 of the MPS VI cats. Twelve unaffected cats underwent complete examinations for comparison purposes. RESULTS: No cardiovascular abnormalities were noted on physical examination. Measured ECG intervals were normal in affected cats; however, sinus arrhythmia was noted more frequently than in the unaffected cats. Significant echocardiographic abnormalities included aortic valve thickening, regurgitation and aortic root dilation. Significant mitral valve thickening was also noted. The severity of changes increased in older affected cats. CONCLUSION: As affected animals increased in age, more cardiac abnormalities were found with increasing severity. Significant lesions included the mitral and aortic valves and ascending aorta, but myocardial changes were not recognized. MPS I and MPS VI cats have similar cardiovascular findings to those seen in children and constitute important models for testing new MPS therapies.

Enhancement of residual arylsulfatase B activity in feline mucopolysaccharidosis VI by thiol-induced subunit association.

The molecular pathology of the deficient arylsulfatase B activity in feline mucopolysaccharidosis (MPS) VI was investigated. Compared with the highly purified normal feline hepatic enzyme, the purified MPS VI residual activity had a 100-fold higher Michaelis constant (K(m)), an altered electrophoretic mobility, half the apparent native molecular weight, and markedly decreased thermo-, cryo-, and pH stabilities. Molecular weight and alkylation studies were consistent with the normal enzyme being a homodimer and the residual MPS VI enzyme a monomer. When incubated with various sulfhydryl reagents, the residual specific activity was enhanced several-fold, whereas the activity of the purified normal enzyme was un-affected or slightly inhibited. In the presence of dithiothreitol (DTT) and cysteamine, a lysosomotropic aminothiol, the residual activity had an electrophoretic mobility and native molecular weight similar to those of the normal feline enzyme. These findings suggested that the monomeric residual enzyme was dimerized in the presence of thiol-reducing agents. To determine if thiol-induced subunit association could therapeutically increase the residual activity and degrade the accumulated dermatan sulfate, in vitro and in vivo experiments were undertaken. When 2 mM DTT or cysteamine was incubated with heparinized whole blood from an MPS VI cat, the leukocyte residual arylsulfatase B activity increased 11- and 20-fold, respectively, and the accumulated dermatan sulfate was degraded in the presence of both thiol reagents. Intravenous administration of DTT (50 mg/kg) effected an immediate, but transient, increase in leukocyte residual activity; however, the substrate levels were not significantly decreased. In contrast, intravenous administration of cysteamine (15 mg/kg) increased leukocyte residual activity more than sixfold 30 min postinfusion; concomitantly, the leukocyte substrate was decreased to 35% of the initial level immediately after infusion and to about 45% of preinfusion values during the 120-min period studied. These results suggest that the defective residual activity in feline MPS VI can be therapeutically manipulated by thiol-induced subunit association. Furthermore, this animal analog provides a prototype for the investigation of human inborn errors of metabolism resulting from enzymatic defects that might be amenable to enzyme manipulation therapy.

Arylsulfatase B activities and glycosaminoglycan levels in retrovirally transduced mucopolysaccharidosis type VI cells. Prospects for gene therapy.

Mucopolysacchariodosis type VI (MPS VI) is the lysosomal storage disorder caused by the deficient activity of arylsulfatase B (ASB; N-acetylgalactosamine 4-sulfatase) and the subsequent accumulation of the glycosaminoglycan (GAG), dermatan sulfate. In this study, a retroviral vector containing the full-length human ASB cDNA was constructed and used to transduce skin fibroblasts, chondrocytes, and bone marrow cells from human patients, cats, or rats with MPS VI. The ASB vector expressed high levels of enzymatic activity in each of the cell types tested and, in the case of cat and rat cells, enzymatic expression led to complete normalization of 35SO4 incorporation. In contrast, overexpression of ASB in human MPS VI skin fibroblasts did not lead to metabolic correction. High-level ASB expression was detected for up to eight weeks in transduced MPS VI cat and rat bone marrow cultures, and PCR analysis demonstrated retroviral-mediated gene transfer to approximately 30-50% of the CFU GM-derived colonies. Notably, overexpression of ASB in bone marrow cells led to release of the enzyme into the media and uptake by MPS VI cat and rat skin fibroblasts and/or chondrocytes via the mannose-6-phosphate receptor system, leading to metabolic correction. Thus, these studies provide important rationale for the development of gene therapy for this disorder and lay the frame-work for future in vivo studies in the animal model systems.

Optimized transduction of canine paediatric CD34(+) cells using an MSCV-based bicistronic vector.

We have used a murine MSCV-based bicistronic retroviral vector, containing the common gamma chain (gammac) and enhanced green fluorescent protein (EGFP) cDNAs, to optimize retroviral transduction of canine cells, including an adherent canine thymus fibroblast cell line, Cf2Th, as well as normal canine CD34(+) bone marrow (BM) cells. Both canine cell types were shown to express Ram-1 (the amphotropic retroviral receptor) mRNA. Supernatants containing infectious viruses were produced using both stable (PA317) and transient (Phoenix cells) amphotropic virus producer cell lines. Centrifugation (spinfection) combined with the addition of polybrene produced the highest transduction efficiencies, infecting approximately 75% of Cf2Th cells. An average of 11% of highly enriched canine CD34(+) cells could be transduced in a protocol that utilized spinfection and plates coated with the fibronectin fragment CH-296 (Retronectin). Indirect assays showed the vector-encoded canine gammac cDNA produced a gammac protein that was expressed on the cell surface of transduced cells. This strategy may result in the transduction of sufficient numbers of CD34(+) BM cells to make the treatment of canine X-linked severe combined immunodeficiency and other canine genetic diseases feasible.

Gene therapy ameliorates cardiovascular disease in dogs with mucopolysaccharidosis VII.

BACKGROUND: Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease caused by deficient beta-glucuronidase (GUSB) activity resulting in defective catabolism of glycosaminoglycans (GAGs). Cardiac disease is a major cause of death in MPS VII because of accumulation of GAGs in cardiovascular cells. Manifestations include cardiomyopathy, mitral and aortic valve thickening, and aortic root dilation and may cause death in the early months of life or may be compatible with a fairly normal lifespan. We previously reported that neonatal administration of a retroviral vector (RV) resulted in transduction of hepatocytes, which secreted GUSB into the blood and could be taken up by cells throughout the body. The goal of this study was to evaluate the effect on cardiac disease. METHODS AND RESULTS: Six MPS VII dogs were treated intravenously with an RV-expressing canine GUSB. Echocardiographic parameters, cardiovascular lesions, and biochemical parameters of these dogs were compared with those of normal and untreated MPS VII dogs. CONCLUSIONS: RV-treated dogs were markedly improved compared with untreated MPS VII dogs. Most RV-treated MPS VII dogs had mild or moderate mitral regurgitation at 4 to 5 months after birth, which improved or disappeared when evaluated at 9 to 11 and at 24 months. Similarly, mitral valve thickening present early in some animals disappeared over time, whereas aortic dilation and aortic valve thickening were absent at all times. Both myocardium and aorta had significant levels of GUSB and reduction in GAGs.

Animal model studies of allelism: characterization of arylsulfatase B mutations in homoallelic and heteroallelic (genetic compound) homozygotes with feline mucopolysaccharidosis VI.

The identification of a second structural gene mutation at the feline arylsulfatase B locus (MPS VIb) provided the opportunity to investigate the expression of allelism by characterization of the residual enzymatic activity in feline mucopolysaccharidosis VI, an animal analogue of human Maroteaux-Lamy syndrome. Matings were designed to produce affected homozygotes who were homoallelic for the MPS VIa and MPS VIb mutations or heteroallelic genetic compounds (MPS VIa/VIb). The physicokinetic and immunological properties of the partially purified residual hepatic arylsulfatase B isozymes from the affected homoallelic and heteroallelic cats were compared to those of the normal feline enzyme. The residual hepatic arylsulfatase B activities from the inbred MPS VIa and MPS VIb homozygotes were distinguished by differences in physicokinetic and immunological properties. The newly identified mutant isozyme b had abnormal kinetic properties toward artificial and natural substrates, normal cryo- and thermostabilities, a normal molecular weight and an altered electrophoretic mobility. Polyacrylamide gel electrophoresis demonstrated that the mutant b subunits formed dimers with normal subunits in obligate heterozygotes (MPS VI+/b). In contrast, mutant isozyme a subunits from obligate MPS VIa/+ heterozygotes did not dimerize with the normal subunit, and the mutant and normal isozymes could be separated by anion exchange chromatography and polyacrylamide gel electrophoresis. Characterization of the partially purified residual hepatic arylsulfatase B activity from the heteroallelic homozygotes revealed the presence of both mutant isozymes a and b. The demonstration of two allelic mutations in the feline arylsulfatase B gene documented the occurrence of genetic heterogeneity in feline mucopolysaccharidosis VI and permitted characterization of the enzymatic defect in homoallelic and heteroallelic (genetic compound) homozygotes, providing a model for the study of allelism in human genetic disorders.

Mucopolysaccharidosis VII in a Cat Caused by 2 Adjacent Missense Mutations in the GUSB Gene.

BACKGROUND: Mucopolysaccharidoses (MPS) are common lysosomal storage disorders causing typically progressive skeletal and ocular abnormalities. OBJECTIVES: To describe the clinic features, metabolic profile and a unique mutation in a domestic shorthair (DSH) kitten with MPS VII. ANIMALS: Affected kitten and 80 healthy cats. METHODS: Serum lysosomal enzyme activities and urinary glycosaminoglycan (GAG) accumulation were assessed. Exons of the ß-glucuronidase gene (GUSB) were sequenced from genomic DNA and genotyping was conducted. RESULTS: A 3-month-old DSH cat was presented for stunted growth, paresis, facial dysmorphia, multiple skeletal deformities, and corneal opacities. Evaluation of blood smears disclosed metachromatic granules in leukocytes and a urinary mucopolysaccharide spot test was positive. The proband had no GUSB activity but normal or increased activities for other lysosomal enzymes. Sequencing of the GUSB gene from the proband and comparison to the sequence of 2 healthy cats and the published feline genome sequence demonstrated 2 unique single base transitions (c.1421T>G and c.1424C>T) in exon 9, altering 2 adjacent codons (p.Ser475Ala and p.Arg476Trp). These amino acid changes are in a highly conserved domain of the GUSB protein and nontolerable to maintain function. Moreover, the p.Arg476Trp mutation previously has been identified in human patients. None of the other clinically healthy cats had these mutations. CONCLUSIONS AND CLINIC IMPORTANCE: The diagnostic approach to MPS disorders is delineated. This is only the second mutation known to cause MPS VII in cats. Similarly, 2 different mutations have been described in MPS VII dogs, thereby showing the molecular heterogeneity of MPS VII in companion animals.

Retrovirus vector-mediated correction and cross-correction of lysosomal alpha-mannosidase deficiency in human and feline fibroblasts.

Lysosomal alpha-mannosidase (EC 3.2.1.24) is an exoglycosidase in the glycoprotein degradation pathway. A deficiency of this enzyme causes the lysosomal storage disease alpha-mannosidosis. Retrovirus vector transfer of a new human alpha-mannosidase cDNA resulted in high-level expression of alpha-mannosidase enzymatic activity in deficient human and feline fibroblasts. The expressed alpha-mannosidase had the same biochemical properties (thermal stability, pH profile, inhibitor/activator sensitivity) as the native enzyme expressed in normal cells. The transferred enzyme colocalized with a control lysosomal hydrolase in cell fractionation experiments. The vector-encoded enzyme also was released at high levels from the corrected cells, and was taken up by untreated mutant cells via the mannose 6-phosphate receptor-mediated endocytic pathway (cross-correction). It is envisioned that genetic correction of a subset of cells (e.g., hematopoietic stem cells) in patients will provide a source of corrective enzyme for other affected tissues in this multisystem disease. Development of a vector expressing high levels of alpha-mannosidase that cross-corrects mutant cells will enable somatic gene transfer experiments in the cat model of human alpha-mannosidosis.

Neonatal intramuscular injection with recombinant adeno-associated virus results in prolonged beta-glucuronidase expression in situ and correction of liver pathology in mucopolysaccharidosis type VII mice.

For many metabolic diseases, early correction of the inherited deficiency is required to prevent long-term sequelae. We examined the ability of adeno-associated virus (AAV) to mediate efficient gene transfer during the neonatal period in mice with the lysosomal storage disease mucopolysaccharidosis type VII (MPS VII). Quadriceps of newborn MPS VII mice were injected with an AAV vector containing human beta-glucuronidase (GUSB) cDNA. High-level intramuscular GUSB expression was seen as early as 2 weeks of age, and persisted for at least 16 weeks with no reduction in activity. In addition, GUSB activity was detected in both liver and spleen at later time points. The level of GUSB activity resulted in a significant reduction in lysosomal storage in the liver and a minimal reduction in the spleen at 16 weeks. However, the temporal and spatial pattern of hepatic GUSB activity, coupled with the presence of GUSB cDNA in liver sections, suggests that hematogenous dissemination of virus at the time of injection led to gene transfer to hepatic cells. These results demonstrate that AAV vectors can successfully infect neonatal muscle and persist through the rapid growth phase following birth. However, GUSB secretion from an intramuscular source is inefficient, limiting the therapeutic efficacy of this approach.

Meningiomas in young cats with mucopolysaccharidosis I.

Meningiomas are the most common central nervous system tumor of the cat, with an age at diagnosis of greater than nine years in 73% of the affected animals. Four of seven cats with alpha-L-iduronidase deficient mucopolysaccharidosis I had meningiomas at postmortem examination. All four cats were less than three years of age. The relationship between the meningiomas and the metabolic defect of MPS I is not clear.

Histopathology, electrodiagnostic testing, and magnetic resonance imaging show significant peripheral and central nervous system myelin abnormalities in the cat model of alpha-mannosidosis.

Alpha-mannosidosis is a disease caused by the deficient activity of alpha-mannosidase, a lysosomal hydrolase involved in the degradation of glycoproteins. The disease is characterized by the accumulation of mannose-rich oligosaccharides within lysosomes. The purpose of this study was to characterize the peripheral nervous system (PNS) and central nervous system (CNS) myelin abnormalities in cats from a breeding colony with a uniform mutation in the gene encoding alpha-mannosidase. Three affected cats and 3 normal cats from 2 litters were examined weekly from 4 to 18 wk of age. Progressively worsening neurological signs developed in affected cats that included tremors, loss of balance, and nystagmus. In the PNS, affected cats showed slow motor nerve conduction velocity and increased F-wave latency. Single nerve fiber teasing revealed significant demyelination/remyelination in affected cats. Mean G-ratios of nerves showed a significant increase in affected cats compared to normal cats. Magnetic resonance imaging of the CNS revealed diffuse white matter signal abnormalities throughout the brain of affected cats. Quantitative magnetization transfer imaging showed a 8%-16% decrease in the magnetization transfer ratio in brain white matter of affected cats compared to normal cats, consistent with myelin abnormalities. Histology confirmed myelin loss throughout the cerebrum and cerebellum. Thus, histology, electrodiagnostic testing, and magnetic resonance imaging identified significant myelination abnormalities in both the PNS and CNS that have not been described previously in alpha-mannosidosis.

Mixed lymphocyte reactivity in cats.

A method was developed to perform primary one-way mixed lymphocyte cultures (MLC) with cat cells. A polymorphic system of MLC reactivities was found within a cat population. In family studies, alloreactivity segregated as a single genetic locus with codominantly expressed products, designated feline lymphocyte defined (Fld)--although closely linked multigene control remains possible. The relationship of Fld to a putative feline major histocompatibility complex is discussed.

Biochemical, pathological, and clinical response to transplantation of normal bone marrow cells into acid sphingomyelinase-deficient mice.

BACKGROUND: Acid sphingomyelinase knock-out (ASMKO) mice are a model of types A and B Niemann-Pick disease. In the present study, we evaluated whether bone marrow transplantation (BMT) carried out on newborn ASMKO mice could prevent or alter the Niemann-Pick disease phenotype. METHODS: Previous work from our laboratory had shown that ASMKO mice were highly susceptible to irradiation-induced death. Therefore, we preconditioned 1-day-old ASMKO (n=35) mice with a "sublethal" dose of 200 cGy of total body irradiation before BMT. The transplantation effects were then analyzed by biochemical, pathological, and clinical approaches. RESULTS: Engraftment ranging from 7% to 100% was achieved in 97% of the transplanted animals. Growth of the engrafted animals was improved, and their survival was increased (from a mean of 5 months to 9 months). The onset of ataxia also was delayed in most of the engrafted animals. In accordance with these observations, biochemical and pathological analysis revealed significant changes in the transplanted group as compared with nontransplanted animals. Lipid storage was reduced in several organs, and there was evidence of histologic improvement seen throughout the reticuloendothelial system, even in animals that were engrafted as low as 14%. In the central nervous system, lipid storage also was reduced, and the Purkinje cells, which are almost absent in ASMKO mice, were present in certain areas of the transplanted animals cerebella. CONCLUSIONS: These results demonstrated that BMT could alter the pathologic phenotype in ASMKO mice, but that this procedure alone was not sufficient to elicit a complete therapeutic effect.

Bone marrow transplantation in newborn rats with mucopolysaccharidosis type VI: biochemical, pathological, and clinical findings.

BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is the lysosomal storage disorder caused by the deficient activity of arylsulfatase B (ASB). In this study, we evaluated bone marrow transplantation (BMT) for the treatment of MPS VI and the effects of irradiation on the survival and engraftment of bone marrow-transplanted neonatal rats. METHODS: One- to 2-day-old MPS VI rats were injected with normal bone marrow after irradiation with 200, 400, or 800 cGy. Ninety percent of the animals receiving a single dose of 200 cGy (n=30) survived the procedure, whereas irradiation with 400 cGy (n=23) or 800 cGy (n=12) resulted in significant mortality (78% and 100%, respectively). Engraftment was monitored by determining ASB activities in peripheral white blood cells and by Y chromosome in situ hybridization analysis. Fifty-two percent of the animals from the 200-cGy group engrafted for up to 8 months after BMT; among the five animals that survived the 400-cGy dose, all engrafted. In comparison, only 20% of nonirradiated animals engrafted at low levels. Of the 24 engrafted animals that were monitored for 8 months after BMT, clinical and/or radiographic improvements were noted in only one (BMT animal 3). Enzymatic analysis revealed that the ASB activities in the reticuloendothelial organs of this animal, as well as two other engrafted but clinically unimproved animals (BMT animals 1 and 2), were normal or near normal; correspondingly, the glycosaminoglycan levels in these organs were significantly reduced. Consistent with the clinical and biochemical observations, light and electron microscopic findings were more improved in BMT animal 3 as compared with BMT animals 1 and 2, although a reduction of storage was evident in each of these transplant recipients, particularly in the trachea and aorta, two tissues that are characteristic sites of pathology in human patients. CONCLUSIONS: These results indicate that BMT in newborn MPS VI patients may prevent many of the pathological and clinical findings in this disorder, but is likely to have very limited and unpredictable effects on the skeletal abnormalities.

Feline mucopolysaccharidosis VI: General ocular and pigment epithelial pathology.

Feline mucopolysaccharidosis VI (MPS VI) is a recessively inherited deficiency of arylsulfatase B (ASB). In the eye, the disease is expressed by the intracytoplasmic accumulation of vacuolated inclusions. These are present in connective tissue cells in the cornea, conjunctiva, sclera, choroid, and the stroma of the iris and ciliary body. In the iris and ciliary body epithelia, only the nonpigmented cells of the latter show presence of the disease. In the retinal pigment epithelium (RPE), a spatial and temporal distribution of the disease has been noted. In general, the nonpigmented RPE in the posterior pole is affected to a greater extent earlier in the disease; the peripheral pigmented RPE remains normal. Although hypertrophy of nonpigmented RPE cells causes disarray of the photoreceptor outer segments, their internal disc organization is not disrupted. Normal outer segment renewal rates and the presence of RPE phagosomes suggest that the diseased RPE is still able to function normally.

Glycosaminoglycan and collagen metabolism in arylsulfatase B-deficient retinal pigment epithelium in vitro.

Regional differences in retinal pigment epithelial (RPE) cell glycosaminoglycan (GAG) and collagen metabolism were studied using cells obtained from normal cats and those with deficient activity of arylsulfatase B (ASB), a lysosomal enzyme involved in GAG catabolism. Control and ASB-deficient RPE cultures initiated from superior equatorial (superior) and inferior equatorial (inferior) regions of the eye were radiolabeled for 72 hr with 35SO4, and GAGs from the media and cell layers were analyzed separately. In ASB-deficient RPE, there was an accumulation of dermatan/chondroitin sulfate in the cell layer of cultures initiated from the superior region of the eye but not in those initiated from the inferior region. This agrees with previous in situ and in vitro morphologic observations that accumulation of inclusions in ASB-deficient RPE was greater in the superior region of the eye than in the inferior region. By contrast, media from ASB-deficient cultures initiated from the inferior region of the eye contained much higher levels of radiolabeled dermatan/chondroitin sulfate than ASB-deficient cultures from the superior region or normal cultures. Increased GAG content in the media may result from increased secretion of proteoglycans, increased turnover of cell surface or extracellular matrix components, or extrusion of lysosomal contents. These results indicate that one or more of these mechanisms vary regionally throughout the eye in the RPE of ASB-deficient animals. Collagen production was determined in normal and ASB-deficient RPE cultures. In normal RPE, no differences in collagen synthesis were noted between the inferior and superior regions.(ABSTRACT TRUNCATED AT 250 WORDS)