RESUMO
In utero ethanol exposure causes fetal alcohol spectrum disorders, associated with reduced brain plasticity; the mechanisms of these effects are not well understood, particularly with respect to glial involvement. Astrocytes release factors that modulate neurite outgrowth. We explored the hypothesis that ethanol inhibits neurite outgrowth by increasing the levels of inhibitory chondroitin sulfate proteoglycans (CSPGs) in astrocytes. Astrocyte treatment with ethanol inhibited the activity of arylsulfatase B (ARSB), the enzyme that removes sulfate groups from chondroitin-4-sulfate (C4S) and triggers the degradation of C4S, increased total sulfated glycosaminoglycans (GAGs), C4S, and neurocan core-protein content and inhibited neurite outgrowth in neurons cocultured with ethanol-treated astrocytes in vitro, effects reversed by treatment with recombinant ARSB. Ethanol also inhibited ARSB activity and increased sulfate GAG and neurocan levels in the developing hippocampus after in vivo ethanol exposure. ARSB silencing increased the levels of sulfated GAGs, C4S, and neurocan in astrocytes and inhibited neurite outgrowth in cocultured neurons, indicating that ARSB activity directly regulates C4S and affects neurocan expression. In summary, this study reports two major findings: ARSB modulates sulfated GAG and neurocan levels in astrocytes and astrocyte-mediated neurite outgrowth in cocultured neurons; and ethanol inhibits the activity of ARSB, increases sulfated GAG, C4S, and neurocan levels, and thereby inhibits astrocyte-mediated neurite outgrowth. An unscheduled increase in CSPGs in the developing brain may lead to altered brain connectivity and to premature decrease in neuronal plasticity and therefore represents a novel mechanism by which ethanol can exert its neurodevelopmental effects.
Assuntos
Astrócitos/efeitos dos fármacos , Proteoglicanas de Sulfatos de Condroitina/efeitos dos fármacos , Sulfatos de Condroitina/metabolismo , Etanol/farmacologia , N-Acetilgalactosamina-4-Sulfatase/farmacologia , Neuritos/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Células Cultivadas , Glicosaminoglicanos/metabolismo , Neuritos/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Neurônios/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Bacterial chondroitinase ABC (ChaseABC) has been used to remove the inhibitory chondroitin sulfate chains from chondroitin sulfate proteoglycans to improve regeneration after rodent spinal cord injury. We hypothesized that the mammalian enzyme arylsulfatase B (ARSB) would also enhance recovery after mouse spinal cord injury. Application of the mammalian enzyme would be an attractive alternative to ChaseABC because of its more robust chemical stability and reduced immunogenicity. A one-time injection of human ARSB into injured mouse spinal cord eliminated immunoreactivity for chondroitin sulfates within five days, and up to 9 weeks after injury. After a moderate spinal cord injury, we observed improvements of locomotor recovery assessed by the Basso Mouse Scale (BMS) in ARSB treated mice, compared to the buffer-treated control group, at 6 weeks after injection. After a severe spinal cord injury, mice injected with equivalent units of ARSB or ChaseABC improved similarly and both groups achieved significantly more locomotor recovery than the buffer-treated control mice. Serotonin and tyrosine hydroxylase immunoreactive axons were more extensively present in mouse spinal cords treated with ARSB and ChaseABC, and the immunoreactive axons penetrated further beyond the injury site in ARSB or ChaseABC treated mice than in control mice. These results indicate that mammalian ARSB improves functional recovery after CNS injury. The structural/molecular mechanisms underlying the observed functional improvement remain to be elucidated.
Assuntos
Locomoção/efeitos dos fármacos , N-Acetilgalactosamina-4-Sulfatase/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/fisiopatologia , Animais , Proteínas de Bactérias/farmacologia , Condroitina ABC Liase/farmacologia , Sulfatos de Condroitina/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Proteínas Recombinantes/farmacologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Degenerative joint changes have been reported in human mucopolysaccharidosis VI (MPS VI) and are a prominent feature of feline MPS VI. Joint disease has proven refractory to intravenous enzyme replacement therapy (ERT) in the MPS VI cat because enzyme is unable to reach cells in cartilage. In this study, enzyme was infused directly into the intraarticular space to determine whether joint tissues are able to respond to replacement enzyme. Clearance of glycosaminoglycans from chondrocytes was observed at a dose of 10 microg recombinant human N-acetylgalactosamine-4-sulfatase (rh4S), but greater clearance was observed with higher doses. The chondrocytes at the articular surface were cleared preferentially. Lysosomal vacuolation in cruciate ligament and synovial cells also decreased upon addition of rh4S. One month after injection of rh4S, a slight reaccumulation of storage was observed at the surface of the joint, but extensive reaccumulation was observed 2 mo after injection. These results indicate that by bypassing the synovium using intraarticular ERT, significant reduction in storage material in joint tissues can be achieved. Localized ERT in the joint space provides a mechanism for delivering enzyme directly to the articular cartilage and a potential therapy for joint pathology in MPS VI.
Assuntos
Gatos , Artropatias , Mucopolissacaridose VI , N-Acetilgalactosamina-4-Sulfatase/uso terapêutico , Animais , Ligamento Cruzado Anterior/citologia , Ligamento Cruzado Anterior/efeitos dos fármacos , Ligamento Cruzado Anterior/metabolismo , Cartilagem Articular/citologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Feminino , Humanos , Artropatias/etiologia , Artropatias/patologia , Artropatias/terapia , Artropatias/veterinária , Articulações/anatomia & histologia , Articulações/patologia , Masculino , Mucopolissacaridose VI/complicações , Mucopolissacaridose VI/patologia , Mucopolissacaridose VI/terapia , Mucopolissacaridose VI/veterinária , N-Acetilgalactosamina-4-Sulfatase/administração & dosagem , N-Acetilgalactosamina-4-Sulfatase/genética , N-Acetilgalactosamina-4-Sulfatase/farmacologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Membrana Sinovial/citologia , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismoRESUMO
OBJECTIVES: To evaluate the safety and efficacy of weekly treatment with human recombinant N-acetylgalactosamine 4-sulfatase (rhASB) in humans with mucopolysaccharidosis type VI (MPS VI). STUDY DESIGN: An ongoing Phase I/II, randomized, two-dose, double-blind study. Patients were randomized to weekly infusions of either high (1.0 mg/kg) or low (0.2 mg/kg) doses of rhASB. Six patients (3 male, 3 female; age 7-16 years) completed at least 24 weeks of treatment, five of this group have completed at least 48 weeks. RESULTS: No drug-related serious adverse events, significant laboratory abnormalities, or allergic reactions were observed in the study. The high-dose group experienced a more rapid and larger relative reduction in urinary glycosaminoglycan that was sustained through week 48. Improvements in the 6-minute walk test were observed in all patients with dramatic gains in those walking <100 meters at baseline. Shoulder range of motion improved in all patients at week 48 and joint pain improved in patients with significant pain at baseline. CONCLUSIONS: rhASB treatment was well-tolerated and reduced lysosomal storage as evidenced by a dose-dependent reduction in urinary glycosaminoglycan. Clinical responses were present in all patients, but the largest gains occurred in patients with advanced disease receiving high-dose rhASB.
Assuntos
Mucopolissacaridose VI/tratamento farmacológico , N-Acetilgalactosamina-4-Sulfatase/uso terapêutico , Adolescente , Criança , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Humanos , Masculino , N-Acetilgalactosamina-4-Sulfatase/efeitos adversos , N-Acetilgalactosamina-4-Sulfatase/farmacologia , Proteínas Recombinantes , Estatísticas não ParamétricasRESUMO
This study evaluates the immunological response following weekly 2h infusions of recombinant human N-acetylgalactosamine 4-sulfatase (rh4S) in Mucopolysaccharidosis VI (MPS VI) cats. The results of three trials (Trial "A": 9 month duration with onset at 3-5 months of age, n = 5; and Trials "B" and "C": 6 month duration starting at birth, n = 9) were compared. No detrimental effects were noted throughout Trials B and C. Temporary hypersensitivity reactions (e.g., vomiting, diarrhoea) occurred in four cats in Trial A and were alleviated by increasing the dose of antihistamine premedication and the duration of infusion. All cats in Trial A developed antibodies to rh4S (range of final titres: 1041-134,931). All cats treated from birth showed negligible titres (range: < 50-598). In vitro inhibition of rh4S activity (up to 47%) was demonstrated with plasma from four cats with elevated titres. Significant reduction of urinary glycosaminoglycan concentration in all cats indicated the ability of rh4S to metabolize stored substrates regardless of the presence of circulating antibodies. Similarly, lysosomal storage in reticuloendothelial cells and fibroblasts of kidney interstistium, dura and skin was reduced in all cats irrespective of their antibody titre although cats with elevated titre had less beneficial effect on cardiovascular tissues (aorta smooth muscle cells, heart valve fibroblasts). Overall improvement in the disease condition (at physical, neurological, and skeletal levels) was most pronounced for cats treated from birth compared with cats treated at a later age.