RESUMO
Metachromatic leukodystrophy (MLD) is a rare lysosomal sphingolipid storage disorder, caused by a deficiency of arylsulfatase A (ASA). It is inherited in an autosomal recessive way, among Caucasians three causing alleles are frequent. Demyelination is the hallmark of MLD. Interest in the disease has increased as therapeutic options such as stem cell transplantation, enzyme replacement and gene therapy are topics of current research. A late-infantile (onset before 3 years of age), a juvenile form (onset before 16 years) and an adult form are usually distinguished. Rapid motor decline is typical for the first and also the second forms, the second may be preceded by cognitive and behavioural problems, which mainly characterize the adult form. There is evidence for a genotype-phenotype correlation: patients homozygous for alleles which do not allow the expression of any enzyme activity (null-allele) suffer from the late infantile form; heterozygosity for a null allele and a non-null allele are more associated with the juvenile form and homozygosity for non-null alleles is more frequent in the most attenuated adult onset form.
Assuntos
Leucodistrofia Metacromática , Estudos de Associação Genética , Humanos , Leucodistrofia Metacromática/diagnóstico , Leucodistrofia Metacromática/genética , Leucodistrofia Metacromática/fisiopatologia , Leucodistrofia Metacromática/terapia , Diagnóstico Pré-Natal , Transplante de Células-TroncoRESUMO
Lysosulfatide is a derivative of the glycosphingolipid sulfatide. It is a major component of high density lipoproteins and was detected in the human brain. Here, we show that lysosulfatide acts as an extracellular signal regulating the migration of a neural precursor cell line (B35 neuroblastoma cells) by rapidly promoting process retraction and cell rounding. These cells express the lysosulfatide receptor S1P3 according to RT-PCR, western blotting and immunocytochemistry, but S1P3 does not mediate the effect since preincubation with three different compounds known to inhibit S1P3 did not block lysosulfatide-induced cell rounding. The signal transduction after stimulation with 3 microM lysosulfatide involves a rapid increase of [Ca2+]i which causes process retraction. This mechanism may be relevant under conditions where neural cells encounter elevated lysosulfatide levels as for example under pathological conditions after breakdown of the blood brain barrier or possibly in the lysosomal sulfatide storage disorder metachromatic leukodystrophy.
Assuntos
Cálcio/fisiologia , Neurônios/fisiologia , Psicosina/análogos & derivados , Células-Tronco/fisiologia , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Forma Celular , Neurônios/citologia , Neurônios/efeitos dos fármacos , Psicosina/farmacologia , Psicosina/fisiologia , Ratos , Receptores de Lisoesfingolipídeo/biossíntese , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacosRESUMO
Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by genetic deficiency of arylsulfatase A (ARSA) enzyme. Failure in catalyzing the degradation of its major substrate, sulfatide (Sulf), in oligodendrocytes and Schwann cells leads to severe demyelination in the peripheral (PNS) and central nervous system (CNS), and early death of MLD patients. The ARSA knockout mice develop a disease that resembles MLD but is milder, without significant demyelination in the PNS and CNS. We showed that adeno-associated virus serotype 5-mediated gene transfer in the brain of ARSA knockout mice reverses Sulf storage and prevents neuropathological abnormalities and neuromotor disabilities when vector injections are performed at a pre-symptomatic stage of disease. Direct injection of viral particles into the brain of ARSA knockout mice at a symptomatic stage results in sustained expression of ARSA, prevention of Sulf storage and neuropathological abnormalities. Despite these significant corrections, the treated mice continue to develop neuromotor disability. We show that more subtle biochemical abnormalities involving gangliosides and galactocerebroside are in fact not corrected.
Assuntos
Encéfalo/enzimologia , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Leucodistrofia Metacromática/terapia , Transdução Genética/métodos , Animais , Química Encefálica , Cerebrosídeo Sulfatase/genética , Cerebrosídeo Sulfatase/metabolismo , Modelos Animais de Doenças , Galactosilceramidas/análise , Galactosilceramidas/metabolismo , Gangliosídeos/análise , Gangliosídeos/metabolismo , Vetores Genéticos/genética , Imuno-Histoquímica , Injeções , Leucodistrofia Metacromática/enzimologia , Camundongos , Camundongos Knockout , Camundongos Mutantes , Atividade Motora , Falha de TratamentoRESUMO
BACKGROUND: P426L and I179S are the two most frequent mutations in juvenile and adult metachromatic leukodystrophy (late-onset MLD), which, in contrast to infantile MLD, show marked phenotypic heterogeneity. OBJECTIVE: To search for genotype-phenotype correlations in late-onset MLD. METHODS: The authors reviewed the clinical course of 22 patients homozygous for mutation P426L vs 20 patients heterozygous for mutation I179S, in which the second arylsulfatase A (ASA) mutation had also been determined. RESULTS: P426L homozygotes principally presented with progressive gait disturbance caused by spastic paraparesis or cerebellar ataxia; mental disturbance was absent or insignificant at the onset of disease but became more apparent as the disease evolved. In contrast, compound heterozygotes for I179S presented with schizophrenia-like behavioral abnormalities, social dysfunction, and mental decline, but motor deficits were scarce. Reduced peripheral nerve conduction velocities and less residual ASA activity were present in P426L homozygotes vs I179S heterozygotes. CONCLUSION: The characteristic clinical differences between homozygous P426L and compound heterozygous I179S patients establish a distinct genotype-phenotype correlation in late-onset metachromatic leukodystrophy.
Assuntos
Cerebrosídeo Sulfatase/genética , Leucodistrofia Metacromática/genética , Fenótipo , Adolescente , Adulto , Cerebrosídeo Sulfatase/metabolismo , Criança , Eletroencefalografia/métodos , Feminino , Genótipo , Humanos , Isoleucina/genética , Leucina/genética , Leucodistrofia Metacromática/fisiopatologia , Imageamento por Ressonância Magnética/métodos , Masculino , Mutação , Condução Nervosa/genética , Condução Nervosa/fisiologia , Prolina/genética , Estatísticas não ParamétricasRESUMO
BACKGROUND: Arylsulfatase A (ASA)-deficient mice are a model for the lysosomal storage disorder metachromatic leukodystrophy. This lipidosis is characterised by the lysosomal accumulation of the sphingolipid sulfatide. Storage of this lipid is associated with progressive demyelination. We have mated ASA-deficient mice with mice heterozygous for a non-functional allele of UDP-galactose:ceramide-galactosyltransferase (CGT). This deficiency is known to lead to a decreased synthesis of galactosylceramide and sulfatide, which should reduce sulfatide storage and improve pathology in ASA-deficient mice. RESULTS: ASA-/- CGT+/- mice, however, showed no detectable decrease in sulfatide storage. Neuronal degeneration of cells in the spiral ganglion of the inner ear, however, was decreased. Behavioural tests showed small but clear improvements of the phenotype in ASA-/- CGT+/- mice. CONCLUSION: Thus the reduction of galactosylceramide and sulfatide biosynthesis by genetic means overall causes modest improvements of pathology.
Assuntos
Cerebrosídeo Sulfatase/genética , N-Acilesfingosina Galactosiltransferase/genética , Uridina Difosfato Galactose/metabolismo , Análise de Variância , Animais , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Encéfalo/patologia , Cruzamento , Cerebrosídeo Sulfatase/deficiência , Cerebrosídeo Sulfatase/metabolismo , Modelos Animais de Doenças , Orelha Interna/metabolismo , Orelha Interna/patologia , Feminino , Galactosilceramidas/metabolismo , Genótipo , Leucodistrofia Metacromática/genética , Leucodistrofia Metacromática/patologia , Leucodistrofia Metacromática/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/fisiologia , N-Acilesfingosina Galactosiltransferase/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Sulfoglicoesfingolipídeos/metabolismo , Fatores de TempoRESUMO
Pluripotency, virtually unlimited self-renewal and amenability to genetic modification make embryonic stem (ES) cells an attractive donor source for cell-mediated gene therapy. In this proof of concept study, we explore whether glial precursors derived from murine ES cells (ESGPs) and engineered to overexpress human arylsulfatase A (hASA) can cross-correct the metabolic defect in an animal model of metachromatic leukodystrophy (MLD). Transfected ES cells showed an up to 30-fold increase in ASA activity. Following in vitro differentiation, high expression of ASA was found in all stages of neural and glial differentiation. hASA-overexpressing ESGPs maintained their ability to differentiate into astrocytes and oligodendrocytes in vitro and in vivo. After transplantation into the brain of neonatal ASA-deficient mice, hASA-overexpressing ESGPs were found to incorporate into a variety of host brain regions. Four weeks after engraftment, immunofluorescence analyses with an antibody to sulfatide revealed a 46.7+/-4.0% reduction of immunoreactive sulfatide deposits in the vicinity of the hASA-positive engrafted cells, thereby significantly extending the rate of sulfatide reduction achieved by the endogenous ASA activity of non-hASA-transfected control cells (21.1+/-5.8%). These findings provide first in vivo evidence that ES cells may serve as a potential donor source for cell-mediated enzyme delivery in storage disorders such as MLD.
Assuntos
Encéfalo/metabolismo , Cerebrosídeo Sulfatase/genética , Células-Tronco Embrionárias/transplante , Terapia Genética/métodos , Leucodistrofia Metacromática/terapia , Sulfoglicoesfingolipídeos/metabolismo , Animais , Western Blotting/métodos , Cerebrosídeo Sulfatase/análise , Cerebrosídeo Sulfatase/metabolismo , Eletroporação , Células-Tronco Embrionárias/metabolismo , Humanos , Imuno-Histoquímica/métodos , Leucodistrofia Metacromática/metabolismo , Camundongos , Camundongos Endogâmicos , Microscopia Confocal , Modelos Animais , Sulfoglicoesfingolipídeos/análise , TransgenesRESUMO
UNLABELLED: Lysosomal storage diseases are clinically heterogeneous with respect to their age of onset, progression of symptoms and the particular organs involved. Varying levels of residual enzyme activity, associated with different defective alleles that cause the respective diseases, are responsible in part for this clinical heterogeneity. In general, the higher the residual enzyme activity, the milder the phenotype. Enzyme activity in severe forms of disease is frequently zero, and in mild forms usually does not exceed approximately 5%. However, the correlation is not so strict as to allow prediction of the phenotype of individual patients. The molecular basis of the different levels of enzyme activity can only be revealed by biochemical investigations of the defective lysosomal proteins. Null alleles may be due to splice-site mutations or deletions. In the case of missense mutations, enzymes frequently fold incorrectly and are retained in the endoplasmic reticulum and subsequently degraded. As these enzymes do not reach the lysosome, they do not provide any functional residual activity. Residual enzyme activity is only observed in cases where the defective enzyme reaches the lysosome and has retained enzymatic activity. Patients carrying the same mutant alleles still show considerable phenotypic variability due to modifying genes and epigenetic factors. None of these has so far been elucidated. However, there are some indications that differences in splicing-factor machinery may influence the phenotypic expression of splice-site mutations and that hormonal modulation of secondary microglial activation in lipidosis may also influence the disease course. CONCLUSION: Phenotypic variability is a frequent phenomenon in lysosomal storage diseases. Residual enzyme activity has been identified as one of the factors influencing the clinical outcome of disease; however, it is obvious that other genetic and epigenetic factors also affect phenotypic variability, particularly in patients with late onset disease.
Assuntos
Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/fisiopatologia , Alelos , DNA Recombinante/genética , Retículo Endoplasmático/metabolismo , Gangliosídeo G(M2)/metabolismo , Gangliosidoses GM2/metabolismo , Gangliosidoses GM2/fisiopatologia , Doença de Gaucher/metabolismo , Doença de Gaucher/fisiopatologia , Humanos , Leucodistrofia Metacromática/metabolismo , Leucodistrofia Metacromática/fisiopatologia , Fenótipo , Mutação Puntual/genética , Doença de Tay-Sachs/metabolismo , Doença de Tay-Sachs/fisiopatologiaRESUMO
Inherited deficiency for the lysosomal enzyme arylsulfatase A (ASA) leads to lysosomal storage of sulfatides and to dramatic demyelination in the CNS of humans (metachromatic leukodystrophy, MLD). As an animal model, ASA(-/-) mice have previously been generated by disruption of the ASA gene and are known to develop lysosomal sulfatide storage similar to that in human MLD, and, moreover, to become deaf because of degeneration of the primary neurons of the auditory pathway. The present study deals with the cellular and topographic distribution of sulfatide storage throughout the CNS of ASA(-/-) mice between a few days and 24 months of age. Sulfatide accumulation was detected on the ultrastructural level and by histochemical staining with alcian blue. Sulfatide storage was found in oligodendroglia and neurons in young mice, and in activated microglia (phagocytes) in adult mice. Neuronal sulfatide storage was most prominent in many nuclei of the medulla oblongata and pons, and in several nuclei of midbrain and forebrain. Sulfatide-storing phagocytes were most frequent in the white matter tracts of aged ASA(-/-) mice, whereas no widespread demyelination was obvious. Loss of neurons was found in two nuclei of the auditory pathway of aged ASA(-/-) mice (ventral cochlear nucleus and nucleus of trapezoid body). The distributional pattern of sulfatide storage throughout the CNS of ASA(-/-) mice largely corresponds to data reported for human MLD. An important difference, however, which remains unexplained at present, is the absence of obvious demyelination from the CNS of ASA(-/-) mice up to the age of 2 years.
Assuntos
Encéfalo/patologia , Leucodistrofia Metacromática/patologia , Lisossomos/ultraestrutura , Sulfoglicoesfingolipídeos/metabolismo , Fatores Etários , Animais , Vias Auditivas/metabolismo , Vias Auditivas/patologia , Vias Auditivas/ultraestrutura , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Cerebrosídeo Sulfatase/deficiência , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Leucodistrofia Metacromática/metabolismo , Masculino , Camundongos , Microglia/metabolismo , Microglia/patologia , Microglia/ultraestrutura , Microscopia Eletrônica de Transmissão , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Oligodendroglia/ultraestrutura , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/ultraestruturaRESUMO
Metachromatic leukodystrophy (MLD) is a lysosomal lipid storage disease caused by arylsulfatase A deficiency. In MLD patients the sphingolipid sulfatide increasingly accumulates leading to progressive demyelination. We have analysed arylsulfatase A-deficient mice, a MLD mouse model, and we show that accumulation of sulfatide is not restricted to the lysosomal compartment but also occurs in myelin itself. Although, this sulfatide storage did not affect the overall composition of most myelin proteins, it specifically caused a severe reduction of MAL. This demonstrates a regulatory link between sulfatide accumulation and MAL expression and indicates the existence of regulatory mechanisms between lipid and myelin protein synthesis in oligodendrocytes. In addition, in cultured renal epithelial cells, sulfatide accumulation diverts MAL to the late endosomal/lysosomal compartment and thus also affects the intracellular distribution of MAL. The specific reduction and mistargeting of MAL protein as a reaction to sulfatide overload may contribute to the pathogenic mechanisms in metachromatic leukodystrophy.
Assuntos
Cerebrosídeo Sulfatase/genética , Leucodistrofia Metacromática/metabolismo , Leucodistrofia Metacromática/fisiopatologia , Microdomínios da Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Proteolipídeos/genética , Proteolipídeos/metabolismo , Animais , Transporte Biológico/fisiologia , Detergentes , Regulação para Baixo , Rim/citologia , Rim/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Bainha de Mielina/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina , Sulfoglicoesfingolipídeos/metabolismoRESUMO
Storage diseases because of lysosomal enzyme deficiencies may be treated by the transplantation of cells that secrete the enzyme which is deficient in patients. One can expect that increasing the amount of secreted enzymes will improve the therapy efficacy. Secretion of lysosomal enzymes can be enhanced by reducing the mannose 6-phosphate receptor involved in the lysosomal sorting of newly synthesized lysosomal enzymes. For this purpose, we have constructed hammerhead ribozymes targeting the mRNA of the large murine mannose 6-phosphate receptor (M6PR300). In vitro ribozymes cleave M6PR300 RNA fragments efficiently with cleavage rates of 69-93% after 3 h of incubation. Ribozymes were cloned into an expression vector in which they are integrated into the VaI adenovirus RNA to increase stability and in which they are transcribed from an RNA polymerase III promoter. These plasmids were transiently transfected into BHK cells to investigate in vivo activity. Two ribozymes reduce efficiently the levels of murine M6PR300 mRNA in transient transfection experiments to 42-45%. This correlates with the reduction of M6PR300 biosynthesis, which is reduced also to 37% of normal. We can also demonstrate that the reduction in M6PR300 is sufficient to increase a lysosomal enzyme secretion.
Assuntos
Terapia Genética/métodos , Doenças por Armazenamento dos Lisossomos/terapia , RNA Catalítico/administração & dosagem , RNA Mensageiro/análise , Receptor IGF Tipo 2/genética , Transfecção/métodos , Animais , Northern Blotting , Linhagem Celular , Cerebrosídeo Sulfatase/metabolismo , Cricetinae , Engenharia Genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Camundongos , Muramidase/genética , Receptor IGF Tipo 2/biossínteseRESUMO
UNLABELLED: Metachromatic leukodystrophy is a lysosomal lipid storage disorder. It is caused by mutations in the gene for arylsulphatase A, an enzyme involved in the degradation of the sphingolipid 3'-O-sulphogalactosylceramide (sulphatide). This membrane lipid can be found in various cell types, but in particularly high concentrations in the myelin of the nervous system. Patients suffer from progressive, finally lethal, demyelination due to accumulation of sulphatide. In the nervous system, lipid storage not only affects oligodendrocytes but also neurons and, in addition, leads to astrogliosis and activation of microglia. At the cellular level, lysosomal sulphatide storage also affects the lipid composition of myelin itself and has consequences for the amount and localization of particular myelin membrane-associated proteins. Here we review data, largely based on an arylsulphatase A knock-out mouse model of metachromatic leukodystrophy. CONCLUSION: The knock-out mouse model of metachromatic leukodystrophy has provided insights into the histopathological and cellular consequences of sulphatide storage.
Assuntos
Leucodistrofia Metacromática/metabolismo , Animais , Arilsulfatases/deficiência , Arilsulfatases/genética , Membrana Celular/metabolismo , Cerebrosídeo Sulfatase , Modelos Animais de Doenças , Imuno-Histoquímica , Leucodistrofia Metacromática/enzimologia , Lipídeos de Membrana/metabolismo , Camundongos , Camundongos Knockout , Glicoproteína Associada a Mielina/metabolismoRESUMO
Arylsulfatase A (ASA) knockout mice represent an animal model for the lysosomal storage disease metachromatic leukodystrophy (MLD). Stem cell gene therapy with bone marrow overexpressing the human ASA cDNA from a retroviral vector resulted in the expression of high enzyme levels in various tissues. Treatment partially reduces sulfatide storage in livers exceeding 18 ng ASA/mg tissue, while complete reduction was observed in livers exceeding 50 ng ASA/mg tissue. This corresponds to about 80% and 200% of normal enzyme activity. Similar values seem to apply for kidney. A partial correction of the lipid metabolism was detectable in the brain where the galactoerebroside/sulfatide ratio, which is diminished in ASA-deficient mice, increased upon treatment. This partial correction was accompanied by amelioration of neuropathology; axonal cross-sectional areas, which are reduced in deficient mice, were significantly increased in the saphenic and sciatic nerve but not in the optic nerve. Behavioral tests suggest some improvement of neuromotor abilities. The gene transfer did not delay the degeneration occurring in the acoustic ganglion of ASA-deficient animals. The limited success of the therapy appears to be due to the requirement of unexpected high levels of ASA for correction of the metabolic defect.
Assuntos
Cerebrosídeo Sulfatase/genética , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Leucodistrofia Metacromática/terapia , Animais , Anticorpos/sangue , Comportamento Animal , Encéfalo/metabolismo , Sistema Nervoso Central/metabolismo , Cerebrosídeo Sulfatase/sangue , Cerebrosídeo Sulfatase/imunologia , Feminino , Vetores Genéticos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Retroviridae/genética , Sulfoglicoesfingolipídeos/metabolismo , Transdução GenéticaRESUMO
The inherited deficiency of arylsulfatase A (ASA) causes lysosomal accumulation of sulfoglycolipids (mainly sulfo-galactosylceramide, S-GalCer ) and leads to metachromatic leukodystrophy in humans. Among visceral organs, kidneys are particularly affected. In the present study, the regional distribution and temporal development of sulfoglycolipid storage in kidneys of ASA-/- mice was investigated histochemically (alcian blue) and ultrastructurally. Furthermore, the sulfoglycolipid storage was examined in kidneys of double-knockout mice, which are incapable of: (a) degrading any sulfolipids (ASA-/-) and (b) synthesizing the major sulfolipid S-GalCer because of deficiency for galactosylceramide synthase (CGT), with the aim to search for additional ASA substrates. In ASA-/- mice, the nephron segments could be ranged in the order of decreasing sulfolipid storage: thin limbs of long-looped nephrons approximately thick ascending limbs > distal convoluted tubules > collecting ducts approximately short thin limbs. Macula densa and proximal tubules were unaffected. In ASA-/-/CGT-/- mice, the long thin limbs and distal convoluted tubules resembled those of ASA-/-/CGT+/+ mice, while the other segments showed less storage. The results suggest that the turnover of sulfolipids in general is highest in the distal nephron except macula densa, and that long thin limbs and distal convoluted tubules are the main sites for turnover of a minor sulfolipid species, which is known to be synthesized in the kidney of CGT-/- mice.
Assuntos
Cerebrosídeo Sulfatase/genética , Galactosiltransferases/genética , Glicolipídeos/metabolismo , Rim/metabolismo , Leucodistrofia Metacromática/genética , Animais , Gangliosídeo Galactosiltransferase , Humanos , Corpos de Inclusão/patologia , Lactente , Rim/patologia , Leucodistrofia Metacromática/metabolismo , Leucodistrofia Metacromática/patologia , Lisossomos/enzimologia , Camundongos , Camundongos Knockout , Néfrons/metabolismoRESUMO
The inherited deficiency of arylsulfatase A (ASA) in humans causes lysosomal accumulation of sulfatides in visceral organs and in the nervous system and leads to wide-spread demyelination (metachromatic leukodystrophy, MLD). ASA-deficient mice have previously been generated by means of targeted gene disruption. In the present study, visceral organs of ASA-deficient mice were investigated. A simple technique for the histochemical detection of accumulated sulfatides was elaborated using pre-embedding staining with alcian blue. The gall bladder, intrahepatic bile ducts, exocrine pancreatic ducts, respiratory epithelium and, with low degree, testicular Sertoli cells, showed sulfolipid storage. The storage pattern in the kidney will be described in a separate publication. Hepatocytes, pancreatic islets, adrenal glands, and gastric epithelium were unaffected. Ultrastructurally, the intralysosomal storage material displayed parallel and concentric lamellar patterns. Apart from some differences, the topographic distribution of the sulfatide storage resembled that in human MLD. In addition to being an animal model of the human disease, the ASA-deficient mouse may be useful for investigating the cell biology of sulfolipids in visceral organs.