i(3)so3-galactosylceramide and Leukodystrophy--Metachromatic

Topics: Cerebroside-Sulfatase; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 22; Dementia; Galactosylceramidase; Genes, Recessive; Humans; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Lysosomes; Mutation; Sulfoglycosphingolipids

Topics: Cerebroside-Sulfatase; Diagnosis, Differential; Genetic Therapy; Humans; Leukodystrophy, Metachromatic; Mutation; Sulfoglycosphingolipids

Topics: Alanine; Aldehydes; Brain; Cysteine; Humans; Kidney; Leukodystrophy, Metachromatic; Sulfatases; Sulfoglycosphingolipids

Topics: Brain Chemistry; Cerebrosides; Female; Gangliosides; Humans; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Pregnancy; Sulfoglycosphingolipids

Topics: Age Factors; Cerebroside-Sulfatase; Enzyme Activation; Fibroblasts; Humans; Leukodystrophy, Metachromatic; Mutation; Sulfatases; Sulfoglycosphingolipids

A review of foreign literature on the pathogenesis and pathomorphology of hereditary diseases of the human nervous system with the myelin involvement is presented. Five forms of leukodystrophies are mainly dealt with: (1) metachromatic, with the defect of their deposition in the form of a metachromatic substance; (2) globoid, with deficiency of galactoceramide beta-galactase enzyme catabolizing cerebrozides and with accumulation of the latter, particularly in the forming "globoid" cells; (3) sudanophilic, with sudanophilic degeneration of the myelin and obscure defect of the enzyme; (4) Pelizaeus-Merzbacher disease with insularly intact myelin; and (5) adrenoleukodystrophy with sudanophilic degeneration of the myelin and involvement of the adrenals. All the forms of leukodystrophies by the time of the onset of the disease are divided into prenatal, late infantile, juvenile, and adult.

Topics: Adolescent; Adult; Central Nervous System; Child; Child, Preschool; Diffuse Cerebral Sclerosis of Schilder; Fetus; Humans; Infant; Infant, Newborn; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Myelin Sheath; Peripheral Nerves; Sulfoglycosphingolipids

Dysmyelination describes an inborn error of metabolism affecting myelinogenesis that causes it to be abnormal, arrested, or delayed. Abiotrophy or myelin as defined by Gowers, due to metabolic failure of the myelin maintenance system, is yet another feature of dysmyelination. In addition to the leukodystrophies, genetically determined conditions such as infantile amaurotic idiocy, hematosidosis, Niemann-Pick's disease and several of the aminoacidopathies are examples of dysmyelinating diseases. In order to reconcile morphological and neurochemical data in these conditions, it is necessary to reexamine a number of pathogenetic hypotheses based on known enzymatic deficiencies, and the interpretation of fragmentary biochemical analyses. The obligatory role of the neuron and axon in myelin formation and maintenance is reviewed. The hypothesis is advanced that gangliosides and their degradative products constitue precursors for the synthesis of the characteristic myelin sphingolipids cerebrosides, sulfatides, and sphingomyelin. Alterations in axoplasmic flow and of ganglioside metabolism must be condidered as important factors in the pathogenesis of dysmyelination.

Topics: Axons; Brain Diseases; Cerebrosides; Galactosidases; Gangliosides; Humans; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Lipidoses; Metabolism, Inborn Errors; Myelin Sheath; Sulfoglycosphingolipids

Galactosylceramides and their sulphates are the main constituents of myelin sheath of the nerve cell. Two genetically determined disorders are the results of an inability to enzymatically hydrolyse these glycolipids. Thus the deficiency of galactosylceramide beta-galactosidase results in globoid cell leucodystrophy and the reduced activity of enzyme, arylsulphatase A is responsible for the disease Metachromatic leucodystrophy. Both these disorders are fatal and are characterized by marked demyelination and severe mental retardation. Since homognenous enzyme preparations of galactosylceramide beta-galactosidase and arylsulphatase A are now available, a possibility of enzyme replacement therapy in globoid and metachromatic leucodystrophies has been discussed.

Topics: beta-Galactosidase; Brain; Cerebroside-Sulfatase; Cerebrosides; Humans; Kidney; Lactose Intolerance; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Liver; Psychosine; Sulfoglycosphingolipids; Sulfurtransferases

Topics: Animals; Arylsulfatases; Brain; Enzyme Activation; Humans; Kidney; Leukodystrophy, Metachromatic; Microsomes; Myelin Sheath; Species Specificity; Sulfatases; Sulfoglycosphingolipids; Sulfurtransferases

Topics: Cells, Cultured; Culture Media; Fibroblasts; Hydrolysis; Leukodystrophy, Metachromatic; Sulfatases; Sulfoglycosphingolipids; Sulfur Radioisotopes

Topics: Adult; Animals; Bone Marrow; Cell Line; Ceramides; Child; Fabry Disease; Fucose; Galactose; Gangliosides; Gaucher Disease; Genetic Variation; Glycolipids; Glycoside Hydrolases; Humans; Leukemia, Myeloid; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Lipid Metabolism, Inborn Errors; Lipidoses; Mice; Niemann-Pick Diseases; Sulfoglycosphingolipids

Topics: Animals; Brain; Carbon Radioisotopes; Cerebrosides; Gangliosides; Humans; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Lipidoses; Nervous System; Niemann-Pick Diseases; Rabbits; Sphingolipids; Sphingomyelins; Sphingosine; Sulfoglycosphingolipids

Use of sulfatide excretion in differentiating MLD/PD-heterozygotes from MLD-patients and PD/PD-homozygotes.. Sulfatide was extracted from urine sediment with chlorotom/methanol (2:1, v/v). The quantity of sulfatide was measured densitometrically (lambda = 580 nm) after thin-layer chromatography. ASA and beta-galactosidase activities were assayed enzymatically.. MLD/PD-heterozygotes excreted sulfatide in the range of 4.8-36.3 nmol/mg lipid (mean +/- SD = 17.8 +/- 10.7), whereas sulfatide in MLD-patients ranged from 74.3-411.6 nmol/mg lipid (mean +/- SD = 184.5 +/- 130.8) and in PD/PD-hormozygotes sulfatide excretion remained in normal range of 0.0-5.9 nmol/mg lipid (mean +/- SD = 1.64 +/- 2.12). ASA activities in these groups were very low or lowered.. The quantitative measurement of sulfatide in urine allows differentiation between MLD/PD-heterozygotes and MLD-heterozygotes, as well as between MLD/PD-heterozygotes with very low ASA activity and MLD-patients or PD/PD-hormozygotes. The quantitative measurement of sulfatide in urine differs between MLD-carriers and controls.

Topics: Adolescent; Adult; Alleles; Cerebroside-Sulfatase; Child; Child, Preschool; Diagnosis, Differential; Female; Heterozygote; Humans; Infant; Leukodystrophy, Metachromatic; Male; Middle Aged; Sulfoglycosphingolipids

Metachromatic leukodystrophy (MLD) is a severe demyelinating, autosomal recessive genetic leukodystrophy. The disease is underpinned by mutations in the arylsulfatase A gene (ARSA), resulting in deficient activity of the arylsulfatase A lysosomal enzyme and consequential accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the brain. Using an ex vivo murine-derived organotypic cerebellar slice culture model, we demonstrate that sulfatide induces demyelination in a concentration-dependent manner. Interestingly, our novel data demonstrate that sulfatide-induced demyelination is underpinned by PARP-1 activation, oligodendrocyte loss, pro-inflammatory cytokine expression, astrogliosis, and microgliosis. Moreover, such sulfatide-induced effects can be attenuated by the treatment with the poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor Olaparib (IC50∼100 nM) suggesting that this small molecule may be neuroprotective and limit toxin-induced demyelination. Our data support the idea that sulfatide is a key driver of demyelination and neuroinflammation in MLD and suggest that PARP-1 inhibitors have therapeutic utility in the sphere of rare demyelinating disease.

Topics: Animals; Cerebroside-Sulfatase; Demyelinating Diseases; Leukodystrophy, Metachromatic; Mice; Neuroinflammatory Diseases; Poly(ADP-ribose) Polymerase Inhibitors; Sulfoglycosphingolipids

A deficiency of human arylsulfatase A (hASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disease characterized by sulfatide accumulation and central nervous system (CNS) demyelination. Efficacy of enzyme replacement therapy (ERT) is increased by genetic engineering of hASA to elevate its activity and transfer across the blood-brain barrier (BBB), respectively. To further improve the enzyme's bioavailability in the CNS, we mutated a cathepsin cleavage hot spot and obtained hASAs with substantially increased half-lives. We then combined the superstabilizing exchange E424A with the activity-promoting triple substitution M202V/T286L/R291N and the ApoEII-tag for BBB transfer in a trimodal modified neoenzyme called SuPerTurbo-ASA. Compared with wild-type hASA, half-life, activity, and M6P-independent uptake were increased more than 7-fold, about 3-fold, and more than 100-fold, respectively. ERT of an MLD-mouse model with immune tolerance to wild-type hASA did not induce antibody formation, indicating absence of novel epitopes. Compared with wild-type hASA, SuPerTurbo-ASA was 8- and 12-fold more efficient in diminishing sulfatide storage of brain and spinal cord. In both tissues, storage was reduced by ∼60%, roughly doubling clearance achieved with a 65-fold higher cumulative dose of wild-type hASA previously. Due to its enhanced therapeutic potential, SuPerTurbo-ASA might be a decisive advancement for ERT and gene therapy of MLD.

Topics: Animals; Brain; Cerebroside-Sulfatase; Humans; Leukodystrophy, Metachromatic; Lysosomal Storage Diseases; Mice; Sulfoglycosphingolipids

Metachromatic leukodystrophy is a neurological lysosomal deposit disease that affects public health despite its low incidence in the population. Currently, few reports are available on pathophysiological events related to enzyme deficiencies and subsequent sulfatide accumulation. This research aims to examine the use of metformin as an alternative treatment to counteract these effects. This was evaluated in human Schwann cells (HSCs) transfected or non-transfected with CRISPR-Cas9, and later treated with sulfatides and metformin. This resulted in transfected HSCs showing a significant increase in cell reactive oxygen species (ROS) production when exposed to 100 µM sulfatides (

Topics: CRISPR-Cas Systems; Humans; Leukodystrophy, Metachromatic; Metformin; Reactive Oxygen Species; Schwann Cells; Sulfoglycosphingolipids

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by an arylsulfatase A (ARSA) deficiency and characterized by severe neurological symptoms resulting from demyelination within the central and peripheral nervous systems. We investigated the feasibility and efficacy of intrathecal administration of a type 9 adeno-associated viral vector encoding ARSA (AAV9/ARSA) for the treatment of 6-week-old MLD model mice, which are presymptomatic, and 1-year-old mice, which exhibit neurological abnormalities. Immunohistochemical analysis following AAV9/ARSA administration showed ARSA expression within the brain, with highest activities in the cerebellum and olfactory bulbs. In mice treated at 1 year, alcian blue staining and quantitative analysis revealed significant decreases in stored sulfatide. Behaviorally, mice treated at 1 year showed no improvement in their ability to traverse narrow balance beams as compared to untreated mice. By contrast, MLD mice treated at 6 weeks showed significant decreases in stored sulfatide throughout the entire brain and improved ability to traverse narrow balance beams. These findings suggest intrathecal administration of an AAV9/ARSA vector is a promising approach to treating genetic diseases of the central nervous system, including MLD, though it may be essential to begin therapy before the onset of neurological symptoms.

Topics: Age Factors; Animals; Cerebellum; Cerebroside-Sulfatase; Dependovirus; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Injections, Spinal; Leukodystrophy, Metachromatic; Mice, Knockout; Spinal Cord; Sulfoglycosphingolipids

Phelan-McDermid syndrome or 22q13.3 deletion syndrome is a rare neurodevelopmental disorder characterized by neonatal hypotonia, severe speech delay, moderate to profound intellectual disability, and minor dysmorphic features. Regression of developmental milestones is often recognized as characteristic of this syndrome. We report a 6-year-old patient with Phelan-McDermid syndrome who presented with rapid neurologic deterioration secondary to metachromatic leukodystrophy due to a mutation of the arylsulfatase A gene (

Topics: Arylsulfatases; Brain; Child; Chromosome Deletion; Chromosome Disorders; Chromosomes, Human, Pair 22; Diagnosis, Differential; Female; Humans; Leukodystrophy, Metachromatic; Magnetic Resonance Spectroscopy; Sulfoglycosphingolipids

An inherited deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) characterized by massive intralysosomal storage of the acidic glycosphingolipid sulfatide and progressive demyelination. Lyso-sulfatide, which differs from sulfatide by the lack of the N-linked fatty acid, also accumulates in MLD and is considered a key driver of pathology although its concentrations are far below sulfatide levels. However, the metabolic origin of lyso-sulfatide is unknown. We show here that ASA-deficient murine macrophages and microglial cells express an endo-N-deacylase that cleaves the N-linked fatty acid from sulfatide. An ASA-deficient astrocytoma cell line devoid of this activity was used to identify the enzyme by overexpressing 13 deacylases with potentially matching substrate specificities. Hydrolysis of sulfatide was detected only in cells overexpressing the enzyme fatty acid amide hydrolase (FAAH). A cell-free assay with recombinant FAAH confirmed the novel role of this enzyme in sulfatide hydrolysis. Consistent with the in vitro data, deletion of FAAH lowered lyso-sulfatide levels in a mouse model of MLD. Regardless of the established cytotoxicity of lyso-sulfatide and the anti-inflammatory effects of FAAH inhibition seen in mouse models of several neurological diseases, genetic inactivation of FAAH did not mitigate, but rather exacerbated the disease phenotype of MLD mice. This unexpected finding was reflected by worsening of rotarod performance, increase of anxiety-related exploratory activity, aggravation of peripheral neuropathy, and reduced life expectancy. Thus, we conclude that FAAH has a protective function in MLD and may represent a novel therapeutic target for treatment of this fatal condition.

Topics: Amidohydrolases; Animals; Cell Line; Cerebroside-Sulfatase; Disease Models, Animal; Female; Leukodystrophy, Metachromatic; Lysosomal Storage Diseases; Mice; Mice, Knockout; Microglia; Primary Cell Culture; Psychosine; Sulfoglycosphingolipids

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed to measure arylsulfatase A (ARSA) activity in leukocytes and dried blood spots (DBS) using deuterated natural sulfatide substrate. These new assays were highly specific and sensitive. Patients with metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD) displayed a clear deficit in the enzymatic activity and could be completely distinguished from normal controls. The leukocyte assay reported here will be important for diagnosing MLD and MSD patients and for monitoring the efficacy of therapeutic treatments. ARSA activity was measured in DBS for the first time without an antibody. This new ARSA DBS assay can serve as a second-tier test following the sulfatide measurement in DBS for newborn screening of MLD. This leads to an elimination of most of the false positives identified by the sulfatide assay.

Topics: Cerebroside-Sulfatase; Chromatography, Liquid; Dried Blood Spot Testing; Humans; Leukocytes; Leukodystrophy, Metachromatic; Molecular Structure; Multiple Sulfatase Deficiency Disease; Sulfoglycosphingolipids; Tandem Mass Spectrometry

The pathological cascade leading from primary storage to neural cell dysfunction and death in metachromatic leukodystrophy (MLD) has been poorly elucidated in human-derived neural cell systems. In the present study, we have modeled the progression of pathological events during the differentiation of patient-specific iPSCs to neuroepithelial progenitor cells (iPSC-NPCs) and mature neurons, astrocytes, and oligodendrocytes at the morphological, molecular, and biochemical level. We showed significant sulfatide accumulation and altered sulfatide composition during the differentiation of MLD iPSC-NPCs into neuronal and glial cells. Changes in sulfatide levels and composition were accompanied by the expansion of the lysosomal compartment, oxidative stress, and apoptosis. The neuronal and glial differentiation capacity of MLD iPSC-NPCs was significantly impaired. We showed delayed appearance and/or reduced levels of oligodendroglial and astroglial markers as well as reduced number of neurons and disorganized neuronal network. Restoration of a functional Arylsulfatase A (ARSA) enzyme in MLD cells using lentiviral-mediated gene transfer normalized sulfatide levels and composition, globally rescuing the pathological phenotype. Our study points to MLD iPSC-derived neural progeny as a useful in vitro model to assess the impact of ARSA deficiency along NPC differentiation into neurons and glial cells. In addition, iPSC-derived neural cultures allowed testing the impact of ARSA reconstitution/overexpression on disease correction and, importantly, on the biology and functional features of human NPCs, with important therapeutic implications.

Topics: Apoptosis; Cell Differentiation; Glycosphingolipids; Humans; Induced Pluripotent Stem Cells; Leukodystrophy, Metachromatic; Lysosomes; Models, Biological; Nerve Degeneration; Neural Stem Cells; Neuroglia; Neurons; Oxidative Stress; Reactive Oxygen Species; Sulfoglycosphingolipids

Invariant NKT (iNKT) cells are innate-like lymphocytes that recognize lipid Ags presented by CD1d. The prototypical Ag, α-galactosylceramide, strongly activates human and mouse iNKT cells, leading to the assumption that iNKT cell physiology in human and mouse is similar. In this article, we report the surprising finding that human, but not mouse, iNKT cells directly recognize myelin-derived sulfatide presented by CD1d. We propose that sulfatide is recognized only by human iNKT cells because of the unique positioning of the 3-

Topics: Animals; Antigen Presentation; Antigens, CD1d; Apolipoproteins E; Cell Line; Cerebroside-Sulfatase; Galactosylceramides; Humans; Leukodystrophy, Metachromatic; Lymphocyte Activation; Mice; Natural Killer T-Cells; Receptors, Antigen, T-Cell; Sulfoglycosphingolipids; Surface Plasmon Resonance; T-Lymphocyte Subsets

Impaired sulfatide catabolism is the primary biochemical insult in patients with the inherited neurodegenerative disease, metachromatic leukodystrophy (MLD), and sulfatide elevation in body fluids is useful in the diagnostic setting. Here we used mass spectrometry to quantify fourteen species of sulfatide, in addition to the deacetylated derivative, lyso-sulfatide, using high pressure liquid chromatography-electrospray ionisation-tandem mass spectrometry in both positive and negative ion mode. A single phase extraction of 0.01 mL of MLD plasma identified all 14 sulfatide species in the positive ion mode but none in the negative ion mode. Interrogation of seven major and seven hydroxylated molecular species, as well as lyso-sulfatide, identified the C18 isoform as the most informative for MLD. The C18 produced a linear response and was below the limit of quantification (<10 pmol mL

Topics: Chromatography, High Pressure Liquid; Enzyme Assays; Humans; Leukodystrophy, Metachromatic; Spectrometry, Mass, Electrospray Ionization; Sulfoglycosphingolipids; Tandem Mass Spectrometry

Allogeneic fetal-derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient-specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene-therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC-derived neural stem cells (NSCs) showing a reliable "NSC signature" is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS-NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a "gold standard" in a side-by-side comparison when validating the phenotype of hiPS-NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS-NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA-overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA-overexpressing iPSC-derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient-specific ARSA-overexpressing hiPS-NSCs may be used in autologous ex vivo gene therapy protocols to provide long-lasting enzymatic supply in MLD-affected brains. Stem Cells Translational Medicine 2017;6:352-368.

Topics: Animals; Cell Differentiation; Cell Line; Cell Movement; Cellular Reprogramming; Cellular Reprogramming Techniques; Cerebroside-Sulfatase; Coculture Techniques; Disease Models, Animal; Enzyme Induction; Gene Expression Regulation, Developmental; Genetic Therapy; Humans; Induced Pluripotent Stem Cells; Leukodystrophy, Metachromatic; Mice, Inbred NOD; Mice, SCID; Nerve Regeneration; Neural Stem Cells; Phenotype; Stem Cell Transplantation; Sulfoglycosphingolipids; Transcriptome

Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder caused by deficiency in arylsulfatase A activity, leading to accumulation of sulfatide substrates. Diagnostic and monitoring procedures include demonstration of reduced arylsulfatase A activity in peripheral blood leukocytes or detection of sulfatides in urine. However, the development of a screening test is challenging because of instability of the enzyme in dried blood spots (DBS), the widespread occurrence of pseudodeficiency alleles, and the lack of available urine samples from newborn screening programs.. We measured individual sulfatide profiles in DBS and dried urine spots (DUS) from MLD patients with LC-MS/MS to identify markers with the discriminatory power to differentiate affected individuals from controls. We also developed a method for converting all sulfatide molecular species into a single species, allowing quantification in positive-ion mode upon derivatization.. In DBS from MLD patients, we found up to 23.2-fold and 5.1-fold differences in total sulfatide concentrations for early- and late-onset MLD, respectively, compared with controls and pseudodeficiencies. Corresponding DUS revealed up to 164-fold and 78-fold differences for early- and late-onset MLD patient samples compared with controls. The use of sulfatides converted to a single species simplified the analysis and increased detection sensitivity in positive-ion mode, providing a second option for sulfatide analysis.. This study of sulfatides in DBS and DUS suggests the feasibility of the mass spectrometry method for newborn screening of MLD and sets the stage for a larger-scale newborn screening pilot study.

Topics: Chromatography, High Pressure Liquid; Dried Blood Spot Testing; Humans; Infant, Newborn; Leukodystrophy, Metachromatic; Mass Spectrometry; Neonatal Screening; Sensitivity and Specificity; Sulfoglycosphingolipids

Topics: Child; Gallbladder; Humans; Immunohistochemistry; Leukodystrophy, Metachromatic; Macrophages; Male; Sulfoglycosphingolipids

Topics: Adult; Cerebroside-Sulfatase; Child, Preschool; Chromatography, High Pressure Liquid; Enzyme Assays; Female; Humans; Kinetics; Leukocytes; Leukodystrophy, Metachromatic; Male; Middle Aged; Reference Standards; Substrate Specificity; Sulfoglycosphingolipids; Tandem Mass Spectrometry

The membrane-bound receptor for platelet-derived growth factor A (PDGFRα) is crucial for controlling the production of oligodendrocytes (OLs) for myelination, but regulation of its activity during OL differentiation is largely unknown. We have examined the effect of increased sulfated content of galactosylceramides (sulfatides) on the regulation of PDGFRα in multipotential neural precursors (NPs) that are deficient in arylsulfatase A (ASA) activity. This enzyme is responsible for the lysosomal hydrolysis of sulfatides. We show that sulfatide accumulation significantly impacts the formation of OLs via deregulation of PDGFRα function. PDGFRα is less associated with detergent-resistant membranes in ASA-deficient cells and showed a significant decrease in AKT phosphorylation. Rescue experiments with ASA showed a normalization of the ratio of long versus short sulfatides, restored PDGFRα levels, corrected its localization to detergent-resistant membranes, increased AKT phosphorylation, and normalized the production of OLs in ASA-deficient NPs. Moreover, our studies identified a novel mechanism that regulates the secretion of PDGFRα in NPs, in glial cells, and in the brain cortex via exosomal shedding. Our study provides a first step in understanding the role of sulfatides in regulating PDGFRα levels in OLs and its impact in myelination.

Topics: Animals; Cells, Cultured; Cerebroside-Sulfatase; Exosomes; Fatty Acids; Leukodystrophy, Metachromatic; Mice, Inbred C57BL; Myelin Sheath; Neural Stem Cells; Neurogenesis; Oligodendroglia; Proteolysis; Receptor, Platelet-Derived Growth Factor alpha; Signal Transduction; Sulfoglycosphingolipids; Transcription, Genetic

Sulfatides are found in brain as components of myelin, oligodendrocytes, and neurons but are also present in various visceral tissues. Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder caused by a deficiency of arylsulfatase A, leading to severe white matter disease due to the accumulation of sulfatides and lysosulfatides. To study the physiological role of sulfatides, accessible and sensitive quantitative methods are required. We developed a sensitive LC/MS/MS method to quantify total sulfatide and lysosulfatide content as well as individual molecular species in urine and plasma from MLD patients and plasma and tissues from an MLD mouse model. Our results demonstrate that the method can quantify a wide range of sulfatide concentrations and can be used to quantify total sulfatide content and levels of individual molecular species of sulfatides in tissues, cells, and body fluids. Even though plasma sulfatides and lysosulfatides would seem attractive candidate biomarkers that could possibly correlate with the severity of MLD and be of use to monitor the effects of therapeutic intervention, our results indicate that it is unlikely that the determination of these storage products in plasma will be useful in this respect.

Topics: Adolescent; Adult; Aged; Animals; Blood Chemical Analysis; Child; Child, Preschool; Chromatography, Liquid; Female; Humans; Infant; Infant, Newborn; Leukodystrophy, Metachromatic; Male; Mice; Middle Aged; Psychosine; Sulfoglycosphingolipids; Tandem Mass Spectrometry; Urinalysis; Young Adult

Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder resulting from a functional deficiency of arylsulfatase A (ARSA), an enzyme that catalyzes desulfation of 3-O-sulfogalactosylceramide (sulfatide). Lack of active ARSA leads to the accumulation of sulfatide in oligodendrocytes, Schwann cells and some neurons and triggers progressive demyelination, the neuropathological hallmark of MLD. Several therapeutic approaches have been explored, including enzyme replacement, autologous hematopoietic stem cell-based gene therapy, intracerebral gene therapy or cell-based gene delivery into the central nervous system (CNS). However, long-term treatment of the blood-brain-barrier protected CNS remains challenging. Here we used MLD patient-derived induced pluripotent stem cells (iPSCs) to generate long-term self-renewing neuroepithelial stem cells and astroglial progenitors for cell-based ARSA replacement. Following transplantation of ARSA-overexpressing precursors into ARSA-deficient mice we observed a significant reduction of sulfatide storage up to a distance of 300 µm from grafted cells. Our data indicate that neural precursors generated via reprogramming from MLD patients can be engineered to ameliorate sulfatide accumulation and may thus serve as autologous cell-based vehicle for continuous ARSA supply in MLD-affected brain tissue.

Topics: Animals; Axons; Brain; Cell Differentiation; Cell Survival; Cell- and Tissue-Based Therapy; Central Nervous System; Cerebroside-Sulfatase; Disease Models, Animal; DNA-Binding Proteins; Gene Expression; Gene Order; Genetic Therapy; Genetic Vectors; Humans; Induced Pluripotent Stem Cells; Lentivirus; Leukodystrophy, Metachromatic; Mice; Mice, Knockout; Neuroglia; Neurons; Sulfoglycosphingolipids; Transduction, Genetic

Metachromatic leukodystrophy (MLD) is an autosomal recessive disease caused by a deficiency in arylsulfatase A (ARSA). However, decreased ARSA activity is also observed in pseudodeficiency (PD). To distinguish between MLD and PD, we performed gene mutation and sulfatide analyses by using dried blood spots (DBSs) from seven Korean individuals who underwent an analysis of ARSA activity. DNA was extracted from DBSs, and PCR-direct sequencing of ARSA was performed. The cDNA obtained was analyzed to confirm a novel mutation. Of the seven subjects, three were confirmed as having MLD, one was confirmed as having MLD-PD, one was confirmed as having PD, and the remaining two were obligate heterozygotes. We verified the novel pathogenic variant c.1107+1delG by performing familial and cDNA analyses. Sulfatide concentrations in DBSs were analyzed and were quantified by using ultra-performance liquid chromatography and tandem mass spectrometry, respectively. Total sulfatide concentration was inversely correlated with ARSA activity (Spearman's coefficient of rank correlation, P=0.929, P=0.0025). The results of this mutational and biochemical study on MLD will increase our understanding of the genetic characteristics of MLD in Koreans.

Topics: Adult; Cerebroside-Sulfatase; Child, Preschool; DNA; DNA Mutational Analysis; Dried Blood Spot Testing; Exons; Genetic Loci; Heterozygote; Humans; Leukodystrophy, Metachromatic; Middle Aged; Polymorphism, Genetic; RNA Splicing; Sulfoglycosphingolipids

Here, we present the design and validation of a new assay for the diagnosis of metachromatic leukodystrophy. The method is highly specific, simple, reproducible, and straightforward. In our spectrophotometric method, the determination of arylsulfatase A (ARSA) activity toward the natural substrate, galactosyl-3-sulfate ceramide (or sulfatide), is performed using neat sulfatide without chemical modification. This confers to the assay high analytical specificity. The hydrolyzed sulfatide is monitored upon inclusion of the colorimetric reagent Azure A. The nonhydrolyzed sulfatide-Azure A is recovered and measured at a wavelength of λ = 650 nm. Thus, ARSA activity toward the sulfatide is obtained by subtracting the nonhydrolyzed sulfatide from the total sulfatide used in the enzyme reaction (sulfatide-Azure A present in a parallel assay performed in the absence of ARSA). Within a clinical context, our method definitely discriminated between healthy subject samples and metachromatic leukodystrophy patient samples, and, therefore, it is suitable for diagnostic applications and for monitoring the efficacy of therapeutic treatments in patients or animal models.

Topics: Animals; Cattle; Cerebroside-Sulfatase; Colorimetry; Enzyme Activation; Humans; Leukodystrophy, Metachromatic; Mice; Spectrophotometry; Sulfoglycosphingolipids

Treatments are being developed for metachromatic leukodystrophy (MLD), suggesting the need for eventual newborn screening. Previous studies have shown that sulfatide molecular species are increased in the urine of MLD patients compared to samples from non-MLD individuals, but there is no data using dried blood spots (DBS), the most common sample available for newborn screening laboratories.. We used ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) to quantify sulfatides in DBS and dried urine spots from 14 MLD patients and 50 non-MLD individuals.. Several sulfatide molecular species were increased in dried urine samples from all MLD samples compared to non-MLD samples. Sulfatides, especially low molecular species, were increased in DBS from MLD patients, but the sulfatide levels were relatively low. There was good separation in sulfatide levels between MLD and non-MLD samples when dried urine spots were used, but not with DBS, because DBS from non-MLD individuals have measurable levels of sulfatides.. Sulfatide accumulation studies in urine, but not in DBS, emerges as the method of choice if newborn screening is to be proposed for MLD.

Topics: Chromatography, Liquid; Dried Blood Spot Testing; Humans; Infant, Newborn; Leukodystrophy, Metachromatic; Sulfoglycosphingolipids; Tandem Mass Spectrometry; Urinalysis

Metachromatic leukodystrophy (MLD) is a genetic autosomal recessive disease caused by a deficiency in arylsulfatase A. Accumulated sulfatides can be detected in the urine and detection of sulfatiduria is a useful test for diagnosis and monitoring. To our knowledge, no studies have explored the accumulation of sulfatides in dried blood spots (DBSs). We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method for measuring sulfatides in DBSs from patients with MLD.. DBSs were eluted with internal standard. After mixing and centrifugation, the organic layer was transferred to a 96-well microplate and dried, then resuspended in methanol/propanol solution. Samples were analyzed on an UPLC system. Total running time was 4 min. Quantification was achieved by multiple reaction monitoring using a tandem mass spectrometer. We evaluated the precision, linearity, and ion suppression of the method and analyzed sulfatide concentrations in DBS specimens from MLD patients (n = 9), pseudodeficiency (PD) patient (n = 1), obligate heterozygotes (OH) (n = 2) and normal controls (n = 124).. In negative-ion mode, sulfatides species subjected to collision-induced dissociation readily fragment to produce an intense ion at m/z 96.8 (HSO4(-)). The precisions of low and high concentration controls ranged from 5.4 to 19.9%. The sulfatides produced linear responses. Molecular species of sulfatides were barely detected in DBSs from normal individuals and the PD-OH group [mean (range), 0.07 (<0.05-0.34) and 0.13 (<0.05-0.22) µg/mL, respectively]. In contrast, the DBSs from MLD patients showed a marked increase in several molecular species of sulfatide [mean (range), 2.02 (1.18-3.89) µg/mL].. Simultaneous detection for sulfatides using UPLC/MS/MS can be successfully applied to DBS analysis. This method provides a fast and effective screening and monitoring tool for the diagnosis and treatment of MLD.

Topics: Case-Control Studies; Child; Child, Preschool; Chromatography, High Pressure Liquid; Dried Blood Spot Testing; Humans; Leukodystrophy, Metachromatic; Linear Models; Reproducibility of Results; Sensitivity and Specificity; Sulfoglycosphingolipids; Tandem Mass Spectrometry

Prediagnostic steps in suspected metachromatic leukodystrophy (MLD) rely on clinical chemical methods other than enzyme assays. We report a new diagnostic method which evaluates changes in the spectrum of molecular types of sulfatides (3-O-sulfogalactosyl ceramides) in MLD urine.. The procedure allows isolation of urinary sulfatides by solid-phase extraction on DEAE-cellulose membranes, transportation of a dry membrane followed by elution and tandem mass spectrometry (MS/MS) analysis in the clinical laboratory. Major sulfatide isoforms are normalized to the least variable component of the spectrum, which is the indigenous C18:0 isoform. This procedure does not require the use of specific internal standards and minimizes errors caused by sample preparation and measurement.. Urinary sulfatides were analyzed in a set of 21 samples from patients affected by sulfatidosis. The combined abundance of the five most elevated isoforms, C22:0, C22:0-OH, C24:0, C24:1-OH, and C24:0-OH sulfatides, was found to give the greatest distinction between MLD-affected patients and a control group.. The method avoids transportation of liquid urine samples and generates stable membrane-bound sulfatide samples that can be stored at ambient temperature. MS/MS sulfatide profiling targeted on the most MLD-representative isoforms is simple with robust results and is suitable for screening.

Topics: Adolescent; Case-Control Studies; Child; Child, Preschool; DEAE-Cellulose; Desiccation; Female; Humans; Infant; Leukodystrophy, Metachromatic; Male; Membranes, Artificial; Middle Aged; Reference Standards; Solid Phase Extraction; Specimen Handling; Sulfoglycosphingolipids; Tandem Mass Spectrometry

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused mainly by mutations in the arylsulfatase A (ARSA) gene. In this manuscript we report sixteen novel mutations identified in the ARSA gene of fifteen unrelated patients affected with MLD. Of these 16 mutations nine were missense mutations (p.L11Q, p.S44P, p.L81P, p.R84L, p.V177D, p.P284S, p.R288S, p.G301R, p.P425S), three were nonsense mutations (p.Q51X, p.Y149X, p.C156X), three were frame shift mutations (c.28delG, c.105C>A+106_124dup, c.189delC) and one was a splice-site mutation (c.1102-2A>G). In addition, three previously reported mutations were identified on an allelic background different from the one in the original reports. Two mutations, p.G309S and p.E312D, were identified on the background of the so-called pseudodeficiency (Pd) allele while previously they were reported alone. On the other hand, mutation p.R311X was identified in two unrelated patients not in cis with the Pd mutations, as previously reported.

Topics: Adolescent; Alleles; Cerebroside-Sulfatase; Child; Child, Preschool; DNA Mutational Analysis; Heterozygote; Homozygote; Humans; Leukodystrophy, Metachromatic; Mutation; Phenotype; Sulfoglycosphingolipids

Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is characterized by an accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). we studied 5/200 cases of MLD and clearly distinguished three clinical forms. One of them presented the juvenile form; two presented the late infantile form; and two other presented the adult form. The Magnetic Resonance Imaging (MRI) of these patients showed a diffuse, bilateral and symmetrical demyelination. The biochemical diagnosis of MLD patients evidencing the low activity of ASA and sulfatide accumulation.. We studied 5/200 MLD patients addressed to us for behavioral abnormalities and progressive mental deterioration. All of them were diagnosed at first by brain MRI evidencing a bilateral demyelination, then the measurement of ASA activity using P-nitrocathecol sulfate as substrate, finally the sulfatiduria was performed using thin-layer chromatography using alpha-naphtol reagent.. In this study, from 200 patients presenting behavioral abnormalities and a progressive mental deterioration, we reported just 2 patients were diagnosed as late-infantile form of MLD. Only1 case presented as the juvenile form; and 2 patients with the adult-type of MLD. The brain magnetic resonance imaging (MRI) of all patients showed characteristic lesions of MLD with extensive demyelination. Biochemical investigations of these patients detected a low level of ASA activity at 0°C and 37°C; the excess of sulfatide in sulfatiduria.. MRI is required to orient the diagnosis of MLD patients; the latter must be confirmed by the biochemical investigations which is based on the measurement of ASA activity and the excess of sulfatide showed in the sulfatiduria.. The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1791578262610232.

Topics: Adult; Biomarkers; Brain; Catechols; Cerebroside-Sulfatase; Child, Preschool; Chromatography, Thin Layer; Female; Humans; Leukodystrophy, Metachromatic; Magnetic Resonance Imaging; Male; Mental Disorders; Phenotype; Predictive Value of Tests; Prognosis; Sulfoglycosphingolipids; Tunisia; Urinalysis

Metachromatic leukodystrophy (MLD) is a rare metabolic disorder leading to demyelination and rapid neurologic deterioration. As therapeutic options evolve, it seems essential to understand and quantify progression of the natural disease. The aim of this study was to assess cerebral volumetric changes in children with MLD in comparison to normal controls and in relation to disease course.. Eighteen patients with late-infantile MLD and 42 typically developing children in the same age range (20-59 months) were analyzed in a cross-sectional study. Patients underwent detailed genetic, biochemical, electrophysiologic, and clinical characterization. Cerebral gray matter (GM) and white matter (WM) volumes were assessed by multispectral segmentation of T1- and T2-weighted MRI. In addition, the demyelinated WM (demyelination load) was automatically quantified in T2-weighted images of the patients, and analyzed in relation to the clinical course.. WM volumes of patients did not differ from controls, although their growth curves were slightly different. GM volumes of patients, however, were on average 10.7% (confidence interval 6.0%-14.9%, p < 0.001) below those of normally developing children. The demyelination load (corrected for total WM volume) increased with disease duration (p < 0.003) and motor deterioration (p < 0.001).. GM volume in patients with MLD is reduced when compared with healthy controls, already at young age. This supports the notion that, beside demyelination, neuronal dysfunction caused by neuronal storage plays an additional role in the disease process. The demyelination load may be a useful noninvasive imaging marker for disease progression and may serve as reference for therapeutic intervention.

Topics: Adolescent; Adult; Aging; Cerebral Cortex; Child; Confidence Intervals; Cross-Sectional Studies; Demyelinating Diseases; Disease Progression; Female; Humans; Image Processing, Computer-Assisted; Leukodystrophy, Metachromatic; Magnetic Resonance Imaging; Male; Middle Aged; Movement Disorders; Myelin Sheath; Neural Conduction; Regression Analysis; Sulfoglycosphingolipids; Young Adult

Sulfatides, a class of acidic glycosphingolipids, are highly expressed in mammalian myelin and in kidney, where they are thought to stabilize neuronal structures and signaling and to influence osmotic stability of renal cells, respectively. Recently, 9-aminoacridine (9-AA) has been introduced as a negative ion matrix that displays high selectivity for low complexity galactosylceramid-I(3)-sulfate sulfatides and that is suitable for quantitative analysis by matrix-assisted desorption/ionization (MALDI) mass spectrometry (MS). Analyzing acidic fractions of lipid extracts and cryosections from kidneys of wild type and arylsulfatase A-deficient (ASA -/-) mice, we demonstrate that 9-AA also enables sensitive on-target analysis as well as imaging of complex lactosylceramide-II(3)-sulfate and gangliotetraosylceramide-II(3), IV(3) bis-sulfate sulfatides by MALDI-TOF/TOF MS. Utilizing the MALDI imaging MS technique, we show differential localization in mouse kidney of (1) sulfatides with identical ceramide anchors, but different glycan-sulfate head groups but also of (2) sulfatides with identical head groups but with different acyl- or sphingoid base moieties. A comparison of MALDI images of renal sulfatides from control and sulfatide storing arylsulfatase A-deficient (ASA -/-) mice revealed relative expression differences, very likely reflecting differences in sulfatide turnover of the various renal cell types. These results establish MALDI imaging MS with 9-AA matrix as a label-free method for spatially resolved ex vivo investigation of the relative turnover of sulfatides in animal models of human glycosphingolipid storage disease.

Topics: Aminacrine; Animals; Cerebroside-Sulfatase; Diagnostic Imaging; Female; Gene Deletion; Humans; Kidney; Leukodystrophy, Metachromatic; Mice; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfoglycosphingolipids

Arylsulfatase A-deficient (ASA(-/-)) mice constitute an animal model for metachromatic leukodystrophy, a lysosomal storage disorder. We had previously examined the behavioural phenotype of these mice, but were unable to distinguish between proper cognitive symptoms and potentially interfering, solely neuromotor impairments. In the present study, T-maze delayed alternation (TMDA) showed that ASA(-/-) mice perform worse than controls already at the age of 6 months in a hippocampus-dependent task that does not require motor proficiency. In addition, long term potentiation (LTP) in the CA1 region of the hippocampus, a cellular correlate of learning and memory, was also impaired in ASA(-/-) mice. Finally, histological analysis of previously unexamined telencephalic and diencephalic structures illustrated sulfatide accumulation in brain areas that are important for cognitive functioning. These include the hippocampus, striatum, internal capsule and diencephalon as well as prefrontal, insular, and motor and somatosensory cortices. Together these data corroborate the usefulness of the model in preclinical evaluations of therapeutic strategies that aim to reverse cognitive defects in the human disease.

Topics: Animals; Brain; Cerebroside-Sulfatase; Disease Models, Animal; Hippocampus; Leukodystrophy, Metachromatic; Long-Term Potentiation; Maze Learning; Mice; Mice, Knockout; Neuronal Plasticity; Sulfoglycosphingolipids

Metachromatic leukodystrophy is a neurodegenerative disease that is characterized by a deficiency of arylsulfatase A, resulting in the accumulation of sulfatide and other lipids in the lysosomal network of affected cells. Accumulation of sulfatide in the nervous system leads to severe impairment of neurological function with a fatal outcome. Prognosis is often poor unless treatment is carried out before the onset of clinical symptoms. Pre-symptomatic detection of affected individuals may be possible with the introduction of newborn screening programs. The ability to accurately predict clinical phenotype and rate of disease progression in asymptomatic individuals will be essential to assist selection of the most appropriate treatment strategy. Biochemical profiling, incorporating the determination of residual enzyme protein/activity using immune-based assays, and metabolite profiling using electrospray ionization-tandem mass spectrometry, was performed on urine and cultured skin fibroblasts from a cohort of patients representing the clinical spectrum of metachromatic leukodystrophy and on unaffected controls. Residual enzyme protein/activity in fibroblasts was able to differentiate unaffected controls, arylsulfatase A pseudo-deficient individuals, pseudo-deficient compound heterozygotes and affected patients. Metachromatic leukodystrophy phenotypes were distinguished by quantification of sulfatide and other secondarily altered lipids in urine and skin fibroblasts; this enabled further differentiation of the late-infantile form of the disorder from the juvenile and adult forms. Prediction of the rate of disease progression for metachromatic leukodystrophy requires a combination of information on genotype, residual arylsulfatase A protein and activity and the measurement of sulfatide and other lipids in urine and cultured skin fibroblasts.

Topics: Adolescent; Adult; Case-Control Studies; Cell Line; Cerebroside-Sulfatase; Child; Child, Preschool; Female; Fibroblasts; Heterozygote; Humans; Infant; Leukodystrophy, Metachromatic; Lysophospholipids; Male; Middle Aged; Monoglycerides; Severity of Illness Index; Skin; Sulfoglycosphingolipids

Metachromatic leukodystrophy (MLD) is a lysosomal disorder caused by arylsulfatase A (ARSA) deficiency. It is classified into three forms according to the age of onset of symptoms (late infantile, juvenile, and adult). We carried out a cross-sectional and retrospective study, which aimed to determine the epidemiological, clinical, and biochemical profile of MLD patients from a national reference center for Inborn Errors of Metabolism in Brazil. Twenty-nine patients (male, 17) agreed to participate in the study (late infantile form: 22; juvenile form: 4; adult form: 1; asymptomatic: 2). Mean ages at onset of symptoms and at biochemical diagnosis were, respectively, 19 and 39 months for late infantile form and 84.7 and 161.2 months for juvenile form. The most frequently reported first clinical symptom/sign of the disease was gait disturbance and other motor abnormalities (72.7%) for late infantile form and behavioral and cognitive alterations (50%) for juvenile form. Leukocyte ARSA activity level did not present significant correlation with the age of onset of symptoms (r = -0.09, p = 0.67). Occipital white matter and basal nuclei abnormalities were not found in patients with the late infantile MLD. Our results suggest that there is a considerable delay between the age of onset of signs and symptoms and the diagnosis of MLD in Brazil. Correlation between ARSA activity and MLD clinical form was not found. Further studies on the epidemiology and natural history of this disease with larger samples are needed, especially now when specific treatments should be available in the near future.

Topics: Adolescent; Age of Onset; Biomarkers; Brazil; Cerebroside-Sulfatase; Child; Child, Preschool; Cross-Sectional Studies; Diagnostic Techniques, Ophthalmological; Disease Progression; Electroencephalography; Eye Diseases; Female; Gait Disorders, Neurologic; Humans; Infant; Leukocytes; Leukodystrophy, Metachromatic; Leukoencephalopathies; Magnetic Resonance Imaging; Male; Mental Disorders; Predictive Value of Tests; Prognosis; Retrospective Studies; Sulfoglycosphingolipids; Time Factors; Young Adult

Neural stem cells appear to be best suited for regenerative therapy in neurological diseases. However, the effects of high levels of potentially toxic substances such as sulfatides--which accumulate in metachromatic leukodystrophy (MLD)--on this regenerative ability are still largely unclear. To start addressing this question, in vitro and in vivo experiments were used to examine the behavior of multipotential neural precursors exposed to abnormally high levels of sulfatides. Following transplantation of dissociated neurospheres into the brain of presymptomatic MLD pups, the majority of donor-derived cells were distributed in a caudal to rostral direction, with higher numbers in the cortex. Most if not all of the donor cells acquired an astroglial phenotype. We found no evidence of oligodendrocyte or neuronal commitment of transplanted cells in long-term-treated MLD mice (e.g. up to 1.5 years of age). This was in line with our in vitro findings of sulfatides blocking oligodendrocyte formation after induction of differentiation in sulfatide-treated epidermal growth factor/fibroblast growth factor responsive neurospheres. Transplanted MLD mice showed an improved arylsulfatase A (ARSA) activity and a significant amelioration of sulfatide metabolism, neurodegeneration and motor-learning/memory deficits. Furthermore, transplanted cells were shown to act as a source of ARSA enzyme that accumulated in endogenous brain cells, indicating the occurrence of enzyme cross-correction between transplanted and host cells. These results provide a first insight into the effect of sulfatides on the stemness properties of neural stem cells and on the effects of the MLD environment on the in vivo expectations of using neural stem cells in cell therapy.

Topics: Animals; Animals, Newborn; Behavior, Animal; Brain; Cell Differentiation; Cell Survival; Cells, Cultured; Cerebroside-Sulfatase; Green Fluorescent Proteins; Humans; Leukodystrophy, Metachromatic; Mice; Mice, Inbred C57BL; Motor Activity; Neurons; Oligodendroglia; Stem Cell Transplantation; Stem Cells; Sulfoglycosphingolipids

3-O-Sulfogalactosylceramides (sulfatides) accumulate in the genetic disease metachromatic leukodystrophy which is due to a defect in the catabolic enzyme, arylsulfatase A. Clinical diagnosis is usually confirmed by in vitro enzymatic deficiency of arylsulfatase A activity. The diagnosis may be complicated because of arylsulfatase A pseudo-deficiencies and another cause of MLD, sphingolipid activator B deficiency. As large quantities of sulfatides can be found in the urine in this disease, sulfatiduria appears as an extremely useful test. As recently enzyme replacement is underway, the quantitative determination, using an internal standard, appears particularly useful as a follow-up. Thus a non-physiological sulfatide was synthesized for this purpose, i.e. 3-O-sulfo-beta-D-C17 galactosylceramide (3-O-Sulfo-D: -Galactosyl-beta1'-->1-N-Heptadecanoyl-D-erythro-Sphingosine). It has been prepared through condensation of an azidosphingosine derivative with a protected D-galactopyranosyltrichloroacetimidate. Reduction of the azide was followed by acylation of a C-17 fatty acid. The key step was achieved by selective sulfation of the desired hydroxyl group on the sugar residue of the galactosylceramide using the stannylene methodology to give a 3'-sulfated beta-galactosyl C-17 ceramide.

Topics: Adult; Cerebroside-Sulfatase; Humans; Leukodystrophy, Metachromatic; Reference Standards; Spectrometry, Mass, Electrospray Ionization; Sulfoglycosphingolipids; Tandem Mass Spectrometry

Metachromatic leukodystrophy (MLD) is a human autosomal recessive lysosomal neurodegenerative disorder that results from the accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). During adolescence and/or adulthood, there are 2 clinical presentations. It may be that of a degenerative disease of the central nervous system with mainly spastic manifestations or a spino-cerebellar ataxia, or that of a psychosis. As several lines of evidence indicate that the psychotic form of MLD could be a model of psychosis, we decided to do a pluridisciplinary study on 11 psycho-cognitive cases involving mental and psychiatric testing, in comparison with 5 adult motor cases, a biochemical study with enzyme assays and quantitative mass spectrometry of urinary sulfatides, so as to determine whether there were biochemical particularities related to the psychotic forms. For quantitative mass spectrometry (MS), a non physiological sulfatide with C17:0 fatty acid was synthesized. The major sulfatide isoforms were present in the 2 clinical forms with the following fatty acids and sphingoid bases: C22:1/d18:1, and /or C22:0/d18:2 (m/z 862.5), C22:0 (OH)/d18:1 (m/z 878,5), C24:0/d18:1 and / or C24:0/C23:1(OH)/d18:2 (m/z 890,3), C24:0 (OH)/d18:1(m/z 906.5). We had shown previously that there were different ASA mutations in the psychiatric adult form (heterozygous I179S) versus the adult motor form (homozygous P426L). We show here that there were no relations with the level of ASA and with the mass spectrometric study of the sulfatide isoforms which were identical in the 2 clinical forms.

Topics: Adolescent; Adult; Age Distribution; Child; Chromatography, Thin Layer; Cognition Disorders; Female; Galactosylceramides; Humans; Leukodystrophy, Metachromatic; Male; Mass Spectrometry; Sulfoglycosphingolipids

An IgM monoclonal antibody, OL-2, was produced by immunizing Lou rats with crude cerebellar membrane fraction. Splenocytes from the rats were fused with a rat myeloma cell line. An antibody secreted by one hybridoma was found to bind to sulfated glycolipids, i.e. sulfatide, seminolipid, sulfolactosylceramide, lysosulfatide and evidenced no binding to neutral sphingoglycolipids such as galactosylceramide, and lactosylceramide, as shown by immunodetection by thin-layer chromatography. In tissue sections, cerebellar white matter and oligodendrocytes were strongly labeled while live; immunocytofluorescence detected both immature and fully mature oligodendrocyte in tissue cultures. The antibody was successfully used to detect urinary sulfatides in metachromatic leukodystrophy and distinguish them from closely migrating other lipids from patients with other neurological diseases.

Topics: Animals; Antibodies, Monoclonal; Blotting, Western; Brain Chemistry; Chromatography, Thin Layer; Epitopes; Humans; Immunohistochemistry; Leukodystrophy, Metachromatic; Rats; Sulfoglycosphingolipids

Enzyme replacement therapy is a treatment option for several lysosomal storage disorders. We reported previously that treatment of a knockout mouse model of the sphingolipid storage disease metachromatic leukodystrophy (MLD) by intravenous injection of recombinant human arylsulfatase A (rhASA) reduces sulfatide storage and improves nervous system pathology and function. Here, we show that treated mice can develop anti-rhASA antibodies, which impede sulfatide clearance without inhibiting enzyme activity. The neutralizing effect of antibodies was reproduced in cell culture models of MLD by demonstrating that mouse immune serum reduces the ability of rhASA to clear sulfatide from cultured ASA-deficient Schwann and kidney cells. We show that reduced clearance is due to an antibody-mediated blockade of mannose 6-phosphate receptor-dependent enzyme uptake, retargeting of rhASA from sulfatide-storing cells to macrophages, intracellular misrouting of rhASA, and reduction of enzyme stability. Induction of immunotolerance to rhASA by transgenic expression of an active site mutant of human ASA restores sulfatide clearance in mice. The data indicate that the influence of non-inhibitory antibodies must be more intensively considered in evaluating the therapeutic efficacy of enzyme replacement in lysosomal storage disorders in general and in patients without cross-reacting material specifically.

Topics: Animals; Antibodies; Binding Sites; Cells, Cultured; Cerebroside-Sulfatase; Genetic Therapy; Humans; Kidney; Leukodystrophy, Metachromatic; Lysosomal Storage Diseases; Mice; Mice, Knockout; Receptor, IGF Type 2; Recombinant Proteins; Sulfoglycosphingolipids

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA) and is characterized by deposition of sulfatide in all organs, particularly the nervous system. Recently, formylglycine-generating enzyme (FGE) was found to be essential for activation of sulfatases. This study examined the utility of FGE co-expression in AAV type 1 vector (AAV1)-mediated gene therapy of ASA knockout (MLD) mice. AAV1-ASA alone or AAV1-ASA and AAV1-FGE were co-injected into a single site of the hippocampus. Enzyme assay and immunohistochemical analysis showed that ASA was detected not only in the injected hemisphere but also in the non-injected hemisphere by 7 months after injection. Level of ASA activity and extent of ASA distribution were significantly enhanced by co-introduction of AAV1-FGE. Marked reductions in sulfatide levels were observed throughout the entire brain. The unexpectedly widespread distribution of ASA may be due to a combination of diffusion in extracellular spaces, transport through axons, and circulation in cerebrospinal fluid. The rotarod test revealed improvement of neurological functions. These results demonstrate that direct injection of AAV1 vectors expressing ASA and FGE represents a highly promising approach with significant implications for the development of clinical protocols for MLD gene therapy.

Topics: Animals; Arylsulfatases; Dependovirus; Disease Models, Animal; Gene Expression Regulation, Enzymologic; Genetic Therapy; Genetic Vectors; Glycine; Humans; Leukodystrophy, Metachromatic; Mice; Sulfoglycosphingolipids

In an effort to develop an encapsulated cell-based system to deliver arylsulfatase A (ARSA) to the central nervous system of metachromatic leukodystrophy (MLD) patients, we engineered C2C12 mouse myoblasts with a retroviral vector containing a full-length human ARSA cDNA and evaluated the efficacy of the recombinant secreted enzyme to revert the MLD phenotype in oligodendrocytes (OL) of the As2-/- mouse model. After transduction, C2C12 cells showed a fifteen-fold increase in intracellular ARSA activity and five-fold increase in ARSA secretion. The secreted hARSA collected from transduced cells encapsulated in polyether-sulfone polymer, was taken up by enzyme-deficient OL derived from MLD mice and normally sorted to the lysosomal compartment, where transferred enzyme reached 80% of physiological levels, restoring the metabolism of sulfatide. To evaluate whether secreted enzyme could restore metabolic function in the brain, encapsulated cells and secreted ARSA were shown to be stable in CSF in vitro. Further, to test cell viability and enzyme release in vivo, encapsulated cells were implanted subcutaneously on the dorsal flank of DBA/2J mice. One month later, all retrieved implants released hARSA at rates similar to unencapsulated cells and contained well preserved myoblasts, demonstrating that encapsulation maintains differentiation of C2C12 cells, stable transgene expression and long-term cell viability in vivo. Thus, these results show the promising potential of developing an ARSA delivery system to the CNS based on the use of a polymer-encapsulated transduced xenogenic cell line for gene therapy of MLD.

Topics: Animals; Arylsulfatases; Capsules; Cell Line; Cell Survival; Disease Models, Animal; Genetic Vectors; Graft Survival; Humans; Leukodystrophy, Metachromatic; Mice; Mice, Knockout; Myoblasts; Nerve Regeneration; Oligodendroglia; Polymers; Sulfoglycosphingolipids; Transduction, Genetic; Transgenes; Transplantation, Heterologous; Treatment Outcome; Up-Regulation

Metachromatic leukodystrophy is a lysosomal storage disorder caused by deficiency in the sulfolipid degrading enzyme arylsulfatase A (ASA). In the absence of a functional ASA gene, 3-O-sulfogalactosylceramide (sulfatide; SGalCer) and other sulfolipids accumulate. The storage is associated with progressive demyelination and various finally lethal neurological symptoms. Lipid storage, however, is not restricted to myelin-producing cells but also occurs in neurons. It is unclear whether neuronal storage contributes to symptoms of the patients. Therefore, we have generated transgenic ASA-deficient [ASA(-/-)] mice overexpressing the sulfatide synthesizing enzymes UDP-galactose:ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST) in neurons to provoke neuronal lipid storage. CGT-transgenic ASA(-/-) [CGT/ASA(-/-)] mice showed an accumulation of C18:0 fatty acid-containing SGalCer in the brain. Histochemically, an increase in sulfolipid storage could be detected in central and peripheral neurons of both CGT/ASA(-/-) and CST/ASA(-/-) mice compared with ASA(-/-) mice. CGT/ASA(-/-) mice developed severe neuromotor coordination deficits and weakness of hindlimbs and forelimbs. Light and electron microscopic analyses demonstrated nerve fiber degeneration in the spinal cord of CGT/ASA(-/-) mice. CGT/ASA(-/-) and, to a lesser extent, young ASA(-/-) mice exhibited cortical hyperexcitability, with recurrent spontaneous cortical EEG discharges lasting 5-15 s. These observations suggest that SGalCer accumulation in neurons contributes to disease phenotype.

Topics: Analysis of Variance; Animals; Behavior, Animal; Cerebral Cortex; Cerebroside-Sulfatase; Disease Models, Animal; Electroencephalography; In Situ Hybridization; Leukodystrophy, Metachromatic; Lipids; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Motor Skills; N-Acylsphingosine Galactosyltransferase; Nerve Degeneration; Neurons; Rats; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spinal Cord; Sulfoglycosphingolipids; Sulfotransferases

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulfatase A (ASA). This results in accumulation of sulfated glycosphingolipids, mainly 3-O-sulfogalactosylceramide (sulfatide), in the nervous system and various other organs. In patients, lipid storage causes a progressive loss of myelin leading to various neurological symptoms. The sulfatide storage pattern in ASA-deficient [ASA(-/-)] mice is comparable to humans, but regrettably, the mice do not mimic the myelin pathology. We reasoned that increasing sulfatide storage in this animal model might provoke demyelination. Therefore, we generated transgenic ASA(-/-) [tg/ASA(-/-)] mice overexpressing the sulfatide-synthesizing enzyme galactose-3-O-sulfotransferase-1 in myelinating cells. Indeed, these tg/ASA(-/-) mice displayed a significant increase in sulfatide storage in brain and peripheral nerves. Mice older than 1 year developed severe neurological symptoms. Nerve conduction velocity was significantly reduced in tg/ASA(-/-) mice because of a peripheral neuropathy characterized by hypomyelinated and demyelinated axons. Inhomogeneous myelin thickness in the corpus callosum, increased frequency of hypomyelinated and demyelinated axons in corpus callosum and optic nerve, and substantially reduced myelin basic protein levels are in accordance with loss of myelin in the CNS. Thus, increasing sulfatide storage in ASA(-/-) mice leads to neurological symptoms and morphological alterations that are reminiscent of human MLD. The approach described here may also be applicable to improve other mouse models of lysosomal as well as nonlysosomal disorders.

Topics: Age Factors; Animals; Cerebroside-Sulfatase; Demyelinating Diseases; Disease Models, Animal; Electromyography; Hindlimb Suspension; Humans; Leukodystrophy, Metachromatic; Lipids; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Motor Activity; Myelin Basic Protein; Myelin Sheath; Neural Conduction; Peripheral Nerves; Rotarod Performance Test; Sciatic Nerve; Spinal Cord; Sulfoglycosphingolipids

This work describes the first successful oligodendrocyte-based cell therapy for presymptomatic arylsulfatase A (ARSA) null neonate mice, a murine model for human metachromatic leukodystrophy (MLD). We found that oligodendrocyte progenitors (OLPs) engrafted and survived into adulthood when transplanted in the neonatal MLD brain. Transplanted cells integrated nondisruptively, did not produce tumors, and survived as proteolipid protein- and MBP-positive postmitotic myelinating oligodendrocytes (OLs) intermingled with endogenous MLD OLs within the adult MLD white matter. Transplanted MLD mice had reduced sulfatide accumulation in the CNS, increased brain ARSA activity, and full prevention of the electrophysiological and motor deficits that characterize untreated MLD mice. Our results provide direct evidence that healthy OLPs can tolerate the neurotoxic accumulation of sulfatides that evolves during the postnatal development of the MLD brain and contribute to OL cell replacement to limit the accumulation of sulfatides and the evolution of CNS defects in this lysosomal storage disease mouse model.

Topics: Animals; Animals, Newborn; Brain Tissue Transplantation; Cell Differentiation; Cell Movement; Cells, Cultured; Cerebroside-Sulfatase; Disease Models, Animal; Graft Survival; Leukodystrophy, Metachromatic; Mice; Mice, Knockout; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin Sheath; Nerve Fibers, Myelinated; Oligodendroglia; Stem Cell Transplantation; Sulfoglycosphingolipids; Treatment Outcome

Glycosphingolipids are located in cell membranes and the brain is especially enriched. We speculated that the subcellular location of glycosphingolipids depends on their fatty acid chain length because their sugar residues are constant, whereas fatty acid chain length can vary within the same molecule. To test this hypothesis we analysed the glycosphingolipid sulfatide, which is highly abundant in myelin and has mostly long fatty acids. We used a negative ion electrospray tandem mass spectrometry precursor ion scan to analyse the molecular species of sulfatide in cultured astrocytes and a mouse model of the human disease metachromatic leukodystrophy. In these arylsulfatase A (ASA)-deficient mice sulfatide accumulates intracellularly in neurons and astrocytes. Immunocytochemistry was also performed on cultured astrocytes and analysed using confocal laser scanning microscopy. Analyses of the molecular species showed that cultured astrocytes contained sulfatide with a predominance of stearic acid (C18), which was located in large intracellular vesicles throughout the cell body and along the processes. The same was seen in ASA-deficient mice, which accumulated a higher proportion (15 mol% compared with 8 mol% in control mice) of sulfatide with stearic acid. We conclude that the major fatty acid composition of sulfatide differs between white and grey matter, with neurons and astrocytes containing mostly short-chain fatty acids with an emphasis on stearic acid. Based on our results, we speculate that the fatty acid chain length of sulfatide might determine its intracellular (short chain) or extracellular (long chain) location and thereby its functions.

Topics: Animals; Astrocytes; Cells, Cultured; Cerebroside-Sulfatase; Fatty Acids; Female; Leukodystrophy, Metachromatic; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Transmission; Neurons; Spectrometry, Mass, Electrospray Ionization; Stearic Acids; Sulfoglycosphingolipids

Metachromatic leukodystrophy (MLD) is an autosomal recessive disease caused by mutations in the gene encoding the lysosomal enzyme arylsulfatase A (ASA). In MLD, accumulation of the substrate, sulfated glycoprotein, in the central and peripheral nervous systems results in progressive motor and mental deterioration. Neural progenitor cells are thought to be useful for cell replacement therapy and for cell-mediated gene therapy in neurodegenerative diseases. In the present study, we examined the feasibility of ex vivo gene therapy for MLD using neural progenitor cells. Neural progenitor cells (neurospheres) were prepared from the striatum of E14 embryo MLD knockout mice or GFP transgenic mice and were transduced with the VSV pseudotyped HIV vector carrying the ASA gene (HIV-ASA). For in vivo study, neurospheres from GFP mice were transduced with HIV-ASA and inoculated into the brain parenchyma of adult MLD mice. HIV vector-transduced progenitor cells retained the potential for differentiation into neurons, astrocytes and oligodendrocytes in vitro. Expression of ASA in neurospheres transduced with HIV-ASA was confirmed by spectrophotometric enzyme assay and Western blotting. In vivo, GFP-positive cells were detectable 1 month after injection. These cells included GFAP- and MAP2-positive cells. Immunohistochemistry using anti-ASA antibody demonstrated localization of ASA in both GFP-positive and -negative cells. Partial clearance of accumulated sulfatide was confirmed in vivo in MLD knockout mice. The present findings suggest that ASA enzyme is released from migrated neurospheres and is able to digest sulfatide in surrounding cells. Our results suggest the potential of genetically engineered neural progenitor cells (neurospheres) for ex vivo therapy in MLD.

Topics: Animals; Brain; Cell Line; Cells, Cultured; Cerebroside-Sulfatase; Female; Genetic Therapy; Genetic Vectors; Glial Fibrillary Acidic Protein; Graft Survival; Green Fluorescent Proteins; HIV-1; Humans; Leukodystrophy, Metachromatic; Mice; Mice, Knockout; Mice, Transgenic; Microtubule-Associated Proteins; Spheroids, Cellular; Stem Cell Transplantation; Stem Cells; Sulfoglycosphingolipids; Transfection; Treatment Outcome

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.

Topics: Animals; Blotting, Western; Brain; Cerebroside-Sulfatase; Electroporation; Embryonic Stem Cells; Genetic Therapy; Humans; Immunohistochemistry; Leukodystrophy, Metachromatic; Mice; Mice, Inbred Strains; Microscopy, Confocal; Models, Animal; Sulfoglycosphingolipids; Transgenes

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.. 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.. Thus the reduction of galactosylceramide and sulfatide biosynthesis by genetic means overall causes modest improvements of pathology.

Topics: Analysis of Variance; Animals; Behavior, Animal; Brain; Breeding; Cerebroside-Sulfatase; Disease Models, Animal; Ear, Inner; Female; Galactosylceramides; Genotype; Leukodystrophy, Metachromatic; Male; Mice; Mice, Knockout; Motor Activity; N-Acylsphingosine Galactosyltransferase; Neurons; Phenotype; Sulfoglycosphingolipids; Time Factors; Uridine Diphosphate Galactose

Metachromatic leukodystrophy (MLD) is a demyelinating lysosomal storage disorder for which new treatments are urgently needed. We previously showed that transplantation of gene-corrected hematopoietic stem progenitor cells (HSPCs) in presymptomatic myeloablated MLD mice prevented disease manifestations. Here we show that HSC gene therapy can reverse neurological deficits and neuropathological damage in affected mice, thus correcting an overt neurological disease. The efficacy of gene therapy was dependent on and proportional to arylsulfatase A (ARSA) overexpression in the microglia progeny of transplanted HSPCs. We demonstrate a widespread enzyme distribution from these cells through the CNS and a robust cross-correction of neurons and glia in vivo. Conversely, a peripheral source of enzyme, established by transplanting ARSA-overexpressing hepatocytes from transgenic donors, failed to effectively deliver the enzyme to the CNS. These results indicate that the recruitment of gene-modified, enzyme-overexpressing microglia makes the enzyme bioavailable to the brain and makes therapeutic efficacy and disease correction attainable. Overall, our data provide a strong rationale for implementing HSPC gene therapy in MLD patients.

Topics: Animals; Behavior, Animal; Cell Differentiation; Cerebroside-Sulfatase; Female; Genetic Therapy; Hematopoietic Stem Cells; Leukodystrophy, Metachromatic; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Neurophysiology; Sulfoglycosphingolipids

Lysosomal exocytosis is a ubiquitously occurring process, which has a physiological role in repair of wounds of the plasma membrane. Lysosomal storage disorders are a group of more than 40 different diseases, which are characterized by intralysosomal storage of various substances. Metachromatic leukodystrophy is a lysosomal disease caused by the deficiency of arylsulfatase A, which results in the storage of the sphingolipid 3-O-sulfogalactosylceramide (sulfatide) in, e.g., oligodendrocytes and distal tubule kidney cells. Here we show that sulfatide storing cultured primary kidney cells of arylsulfatase A deficient mice can undergo calcium induced lysosomal exocytosis and that this results in the delivery of storage material to the culture medium. In metachromatic leukodystrophy extracellular sulfatide has been found in urine and cerebrospinal fluid. Lysosomal exocytosis may explain the presence of sulfatide in these body fluids.

Topics: Animals; Arylsulfatases; Calcium; Cells, Cultured; Exocytosis; Galactosylceramides; Kidney Tubules, Distal; Leukodystrophy, Metachromatic; Lysosomal Storage Diseases; Lysosomes; Mice; Mice, Knockout; Mice, Mutant Strains; Oligodendroglia; Sulfoglycosphingolipids

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. This leads to the accumulation of 3-O-sulfogalactosylceramide, which results in severe demyelination. Here we describe a novel non-sense mutation W124ter and two disease-causing missense mutations E382Q and C500F in arylsulfatase A gene. Another so far unknown allele harbors three sequence alterations: two polymorphisms (N350S, R496H) and a missense mutation (R288H). The R288H substitution and the N350S polymorphism have previously been found on one allele together with a polymorphism in a polyadenylation signal characteristic for the arylsulfatase A pseudodeficiency allele. The R496H has been shown to occur on another allele. The presence of the R288H, N350S, and R496H substitution on one allele in the absence of the polyadenylation site polymorphism shows that this allele has probably arisen by recombination between the nucleotides of codon 350 and 496.

Topics: Alleles; Animals; Blotting, Northern; Cell Line; Cerebroside-Sulfatase; Cricetinae; Leukodystrophy, Metachromatic; Mutagenesis, Site-Directed; Polymorphism, Genetic; Precipitin Tests; Sulfoglycosphingolipids

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.

Topics: Animals; Biological Transport; Cerebroside-Sulfatase; Detergents; Down-Regulation; Kidney; Leukodystrophy, Metachromatic; Lysosomes; Membrane Microdomains; Membrane Proteins; Membrane Transport Proteins; Mice; Mice, Mutant Strains; Myelin and Lymphocyte-Associated Proteolipid Proteins; Myelin Proteins; Myelin Sheath; Proteolipids; Sulfoglycosphingolipids

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.

Topics: Age Factors; Animals; Auditory Pathways; Brain; Cerebroside-Sulfatase; Disease Models, Animal; Female; Humans; Immunohistochemistry; Leukodystrophy, Metachromatic; Lysosomes; Male; Mice; Microglia; Microscopy, Electron, Transmission; Neurons; Oligodendroglia; Spinal Cord; Sulfoglycosphingolipids

Metachromatic leukodystrophy (MLD) is an inherited demyelinating disorder caused by the deficiency of arylsulphatase A (ASA). This defect leads to an accumulation of galactosylceramide I(3)-sulphates (sulphatides) in lysosomes of different tissues. We report on mutations found in a group of nine patients from the Czech and Slovak Republics (former Czechoslovakia). Their diagnosis was confirmed by determination of the activity of arylsulphatase A in leukocytes and by abnormal urinary excretion of sulphatides. All alleles of the patients were identified and eight different mutations were found. They include four novel missense mutations in one infantile (D29N), one juvenile (C294Y), and three adult (C156R, G293S) patients. Four mutations were previously described sequence alterations (459 + 1G > A, G309S, I179S, and P426L). Polymorphisms characteristic for the ASA pseudodeficiency allele were not found in the patients. Substitutions of D29N, C294Y, and G293S in arylsulphatase A caused a severe reduction of enzyme activity in transient expression studies. In contrast, the C156R substitution reduces arylsulphatase A only to 50% of wild type ASA activity. Since no other mutations were found in this patient, the contribution of this mutation to the development of disease remains unclear.

Topics: Adolescent; Adult; Cells, Cultured; Cerebroside-Sulfatase; Child; Child, Preschool; Czech Republic; DNA Primers; Female; Genotype; Humans; Infant; Leukocytes; Leukodystrophy, Metachromatic; Male; Mutagenesis; Mutation; Phenotype; Sequence Analysis, DNA; Slovakia; Sulfoglycosphingolipids; Transfection

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder most often caused by mutations in the sulfatide sulfatase or arylsulfatase A (ASA) gene. This results in the storage of sulfatides in the peripheral and central nervous systems as well as in the kidneys. Patients with MLD exhibit a wide range of clinical features presenting from the late infantile period to adulthood. Testing for this disease is performed on a majority of the patient samples received for diagnostic testing in the authors' laboratory. If low ASA activity is measured, additional testing is required to confirm the diagnosis due to several factors. ASA activity is also low in individuals with multiple sulfatase deficiency and in individuals with copies of the so-called pseudodeficiency (Pd) allele. Due to the high frequency of the common Pd allele, it is possible for individuals, both with and without neurologic disease, to have low ASA activity but not have MLD. Unfortunately, the finding of the Pd mutation by molecular analysis does not rule out a diagnosis of MLD. In a recent 25 month period, this laboratory diagnosed 52 patients with MLD, and of these, 13 patients from 10 families had one or two copies of the Pd mutation. Sequencing of the ASA gene in these 10 families revealed four new mutations in cis with the Pd allele (S43R, R84Q, R311X, and E329R) and two additional new mutations (R299W, C488R). Six patients had previously reported mutations on the Pd background. Thus, a total of 14 mutations have been found to occur in cis with the Pd allele. We estimate that 1-2% of Pd alleles will have a disease-causing mutation, and this complicates the identification of patients and the assignment of risk for a couple when a copy of the Pd allele is detected.

Topics: Adult; Alleles; Cerebroside-Sulfatase; Child; Child, Preschool; Genetic Predisposition to Disease; Heterozygote; Homozygote; Humans; Infant; Leukodystrophy, Metachromatic; Mutation; Pedigree; Polymorphism, Genetic; Sulfoglycosphingolipids

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by the deficiency of arylsulfatase A (ARSA) or saposin B. The majority of mutations identified in patients with MLD are unique within individual families. Here, we report on the novel missense mutations (F247S, D381E, and A469G) and the known mutations "A" allele and P136S in the ARSA gene in three unrelated Ukrainian families with MLD. The mutations F247S and P136S were found in compound heterozygous with the "A" allele in two patients with juvenile onset MLD. The clinical features of the typical patient with genotype D381E/A469G (early onset with very rapid manifestation of disease) suggest the reason to distinguish an early infantile MLD variant.

Topics: Amino Acid Sequence; Base Sequence; Cerebroside-Sulfatase; Child; Child, Preschool; Female; Humans; Infant; Leukodystrophy, Metachromatic; Mutation, Missense; Phenotype; Polymorphism, Restriction Fragment Length; Sequence Analysis, DNA; Sulfoglycosphingolipids; Ukraine

Potential damage of central and peripheral nervous system expressed as micro-organic brain damage (MOBD) was investigated in 27 unrelated heterozygotes with metachromatic leukodystrophy (MLD). Arylsulfatase A (ARSA) was determined in peripheral blood leukocytes and sulfatide excretion was estimated in 24-hour urine collections. Genomic DNA was analyzed for the ARSA pseudodeficiency (PD) allele by a PCR method. Clinical investigations included examination of hyper-reflexia, Babinski reflex, Wechsler Adult Intelligence Scale, Benton test, evoked potentials, and nerve conduction velocity (NCV). In our study, a higher incidence of evident or possible micro-organic brain damage was observed in true MLD/PD and MLD heterozygotes (NO/MLD, where NO means the wild allele) than in controls. On the basis of the Benton test, MOBD was suggested or indicated in 67% of MLD heterozygotes, 50% of MLD/PD heterozygotes, and 26% of controls. In our small group of carriers with MLD and PD mutations, persons NO/MLD(PD) with one wild-type allele did not show MOBD and displayed higher ARSA/beta-galactosidase ratios, unlike true MLD/PD compound heterozygotes who carry MLD-causing mutation in one allele and the ARSA-PD polymorphism in the second. Theoretically, this is a shift from autosomal recessive to autosomal dominant-like inheritance, especially when one cannot exclude the influence of polymorphisms (like ARSA-PD) in the wild allele. Since all psychological tests were age-matched, it can be assumed that the MOBD observed in MLD carriers does not have a progressive character unlike in MLD patients. However, it should be mentioned that MOBD appears to have no overt clinical consequences.

Topics: Adult; beta-Galactosidase; Brain Diseases; Cerebroside-Sulfatase; DNA; Female; Gene Frequency; Genotype; Heterozygote; Humans; Leukodystrophy, Metachromatic; Male; Middle Aged; Neuropsychological Tests; Sulfoglycosphingolipids

Topics: Brain; Brain Chemistry; Cerebroside-Sulfatase; Cerebrosides; Child; Fatal Outcome; Female; Humans; Leukocytes; Leukodystrophy, Metachromatic; Sulfoglycosphingolipids

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.

Topics: Animals; Antibodies; Behavior, Animal; Brain; Central Nervous System; Cerebroside-Sulfatase; Female; Genetic Therapy; Genetic Vectors; Hematopoietic Stem Cell Transplantation; Leukodystrophy, Metachromatic; Liver; Male; Mice; Mice, Knockout; Models, Animal; Retroviridae; Sulfoglycosphingolipids; Transduction, Genetic

Metachromatic leukodystrophy is an inherited disorder characterized by a deficiency of the lysosomal enzyme arylsulfatase A and the subsequent accumulation of sulfatide in neural and visceral tissues. Clinical diagnosis is usually confirmed by in vitro analysis of arylsulfatase A activity, but may be complicated in cases of arylsulfatase A pseudodeficiency and sphingolipid activator protein deficiency. Large quantities of sulfatide can be detected in the urinary sediment of affected individuals and its measurement can aid in diagnosis. A number of complex methods have been described for the measurement of urinary sulfatide excretion. We have developed a rapid, sensitive, and specific mass spectrometric method for determining urinary sulfatide concentration of metachromatic leukodystrophy patients. Sulfatides are extracted from urine and then directly analyzed using electrospray ionization-tandem mass spectrometry. A sulfatide internal standard has been employed for quantification. The assay has demonstrated significant elevations in the concentrations of several hydroxy and nonhydroxy molecular species of sulfatide in the urine of metachromatic leukodystrophy patients compared to age-matched controls. Analysis of urinary sulfatides in arylsulfatase A pseudodeficiency patients showed a mild elevation in some individuals when related to urinary phosphatidylcholine.

Topics: Adolescent; Adult; Child; Child, Preschool; Female; Humans; Leukodystrophy, Metachromatic; Male; Mass Spectrometry; Phosphatidylcholines; Sulfoglycosphingolipids

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.

Topics: Animals; Cerebroside-Sulfatase; Child, Preschool; Disease Models, Animal; Histocytochemistry; Humans; Leukodystrophy, Metachromatic; Lysosomes; Male; Mice; Mice, Knockout; Microscopy, Electron; Sulfoglycosphingolipids; Tissue Distribution; Viscera

Topics: Cerebroside-Sulfatase; Humans; Leukocytes; Leukodystrophy, Metachromatic; Lysosomal Storage Diseases; Sulfoglycosphingolipids; Sulfotransferases

The lysosomal degradation of sphingolipids with short oligosaccharide chains depends on small glycosylated non-enzymatic sphingolipid activator proteins (SAPs, saposins). Four of the five known SAPs, SAP-A, -B, -C and -D, are derived by proteolytic processing from a common precursor protein (SAP-precursor) that is encoded by a gene on chromosome 10 consisting of 15 exons and 14 introns. SAP-B is a non-specific glycolipid binding protein that stimulates in vitro the hydrolysis of about 20 glycolipids by different enzymes. In vivo SAP-B stimulates in particular the degradation of sulphatides by arylsulphatase A. So far, four different point mutations have been identified on the SAP-B domain of the SAP-precursor gene. The mutations result in a loss of mature SAP-B, causing the lysosomal accumulation of sulphatides and other sphingolipids, resulting in variant forms of metachromatic leukodystrophy (MLD). Here we report on a patient with SAP-B deficiency that is caused by a new homoallelic point mutation that has been identified by mRNA and DNA analysis. A 643A > C transversion results in the exchange of asparagine 215 to histidine and eliminates the single glycosylation site of SAP-B. Metabolic labelling experiments showed that the mutation had no effect on the intracellular transport of the encoded precursor to the acidic compartments and its maturation in the patient's cells. All four SAPs (SAP-A to SAP-D) were detectable by immunochemical methods. SAP-B in the patient's cells was found to be slightly less stable than the protein in normal cells and corresponded in size to the deglycosylated form of the wild-type SAP-B. Feeding studies with non-glycosylated SAP-precursor, generating non-glycosylated SAP-B, showed that the loss of the carbohydrate chain reduced the intracellular activity of the protein significantly. The additional structural change of the patient's SAP-B, caused by the change of amino acid 215 from asparagine to histidine, presumably resulted in an almost completely inactive protein.

Topics: Child, Preschool; Female; Fibroblasts; Glycoproteins; Glycosylation; Humans; Leukodystrophy, Metachromatic; Mutation; Saposins; Sphingolipid Activator Proteins; Sulfoglycosphingolipids

A fast and simple method for determination of sulfatides in the urine of patients with metachromatic leukodystrophy (MLD, arylsulfatase A deficiency) has been developed. The procedure consists of two steps: extraction of total urinary lipids by reversed-phase chromatography and their HPTLC separation. Two types of sorbents based on different matrixes were compared, of which the hydroxyethyl methacrylate C-18 type sorbent was found to be superior. Twenty-milliliter aliquots of urine are sufficient for the analysis. The technique is appropriate for simultaneous qualitative identification and semiquantitative densitometric determination and is suitable for routine work. The amount of sulfatides is expressed in relation to sphingomyelin, which copurifies with sulfatides and better reflects the level of membrane lipids in urine than commonly used parameters (creatinine, urine volume, etc.). The ranges were found to be 0.15-0.68 nmol sulfatide/nmol sphingomyelin for control individuals and 3.5-27.2 nmol sulfatide/nmol sphingomyelin for MLD patients. The excretion of sulfatides is pathonognomic for true MLD (due to the accumulation in kidney) and therefore its analysis is important for evaluation of suspected MLD cases including clinically and enzymatically atypical cases. The method is also useful as a complementary analysis for other lipidoses with high excretion of sphingolipids in urine (e.g., Fabry disease).

Topics: Case-Control Studies; Chromatography; Chromatography, Thin Layer; Fabry Disease; Humans; Leukodystrophy, Metachromatic; Lipids; Proteinuria; Sulfoglycosphingolipids

Arylsulfatase A (ARSA) deficiency is the main cause of metachromatic leukodystrophy (MLD), a lysosomal disorder with no specific treatment. In view of the importance of genetic counseling, analyses of mutations and polymorphisms, including the ARSA pseudodeficiency allele, were carried out in 18 unrelated Spanish MLD patients. A systematic search allowed us to identify 100% of the alleles involving 17 different mutations, 12 of which are novel: G32S, L68P, R84W, P94A, G99V, P136S, W193X, H227Y, R288H, G308D, T327I, and IVS6-12C-->G. Two new polymorphisms, 2033C>T and 2059C>T, were identified in intron 6 which, in combination with two polymorphisms previously described (2161C>G and 2213C>G), gave rise to four different haplotypes in the control population. In addition, we also studied polymorphism 842G>T. Linkage disequilibrium was detected between mutations IVS2+1G-->A, D255H, and T327I and specific haplotypes, suggesting a unique origin for these mutations. Moreover, mutation T327I was always associated with the T allele of the new rare variant A210A (893C>T). The distribution of mutation D255H (frequency 19.4%) among patients with different MLD clinical presentation revealed a clear genotype-phenotype correlation paralleling that reported for mutation IVS2+1G-->A (frequency 25%). Among the novel mutations, only P136S and R288H occurred on a background of the ARSA pseudodeficiency allele. Screening 182 normal chromosomes identified a frequency of 8.8% of this allele; moreover, we identified two unrelated subjects with the polyA- mutation in the absence of the N350S mutation, and this infrequent haplotype reinforced the heterogeneity of conditions with ARSA deficiency.

Topics: Adolescent; Adult; Age of Onset; Alleles; Cells, Cultured; Cerebroside-Sulfatase; Child; Child, Preschool; Chromosome Mapping; Chromosomes, Human, Pair 22; DNA Mutational Analysis; Female; Fibroblasts; Gene Frequency; Haplotypes; Humans; Infant; Leukodystrophy, Metachromatic; Male; Mutation; Phenotype; Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Single-Stranded Conformational; Spain; Sulfoglycosphingolipids

A deficiency of the lysosomal enzyme arylsulfatase A (ASA) causes the lysosomal storage disorder metachromatic leukodystrophy (MLD). The diagnosis of MLD is straightforward in cases with deficient leukocyte or fibroblast ASA activity and a typical clinical history. However, several atypical and late-onset forms of MLD have been described. The diagnosis is also complicated by the high frequency of presumably benign polymorphisms at the ASA gene locus that are associated with markedly diminished in vitro ASA activity. Additional diagnostic tools are needed in the clinically and (or) enzymatically atypical cases. Although analyses of urinary sulfatides have been reported to be helpful in the diagnosis of MLD, previously described methods are complex and incompletely characterized and validated. We developed an improved method for determining urinary sulfatides and applied it to a cohort of individuals with MLD. The sulfatides are extracted from urine, separated from glycerol-based lipids by alkaline hydrolysis, isolated by ion-exchange chromatography, and hydrolyzed to galactosylceramide, which is then perbenzoylated and quantified by HPLC. This assay provides excellent resolution of sulfatides from other lipids and good analytical precision. In addition, the urinary sulfatide concentrations of healthy controls (mean +offSD: 0.16 +/- 0.07 nmol/mg creatinine; range: 0.07-0.34; n = 18) are clearly distinguished from those of individuals with MLD (7.6 +/- 6.1 nmol/mg creatine; 1.2-24.2; n = 20).

Topics: Cerebroside-Sulfatase; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Humans; Hydrogen-Ion Concentration; Hydrolysis; Leukodystrophy, Metachromatic; Reference Values; Sensitivity and Specificity; Sulfoglycosphingolipids

Topics: Bone Marrow Transplantation; Cerebroside-Sulfatase; Child, Preschool; Disease Progression; Female; Follow-Up Studies; Humans; Leukodystrophy, Metachromatic; Nervous System; Sulfoglycosphingolipids

We identified a patient suffering from late-infantile metachromatic leukodystrophy (MLD) who has a residual arylsulfatase A (ARSA) activity of about 10%. Fibroblasts of the patient show significant sulfatide degradation activity exceeding that of adult MLD patients. Analysis of the ARSA gene in this patient revealed heterozygosity for two new mutant alleles: in one allele, deletion of C 447 in exon 2 leads to a frameshift and to a premature stop codon at amino acid position 105; in the second allele, a G-->A transition in exon 5 causes a Gly309-->Ser substitution. Transient expression of the mutant Ser309-ARSA resulted in only 13% enzyme activity of that observed in cells expressing normal ARSA. The mutant ARSA is correctly targeted to the lysosomes but is unstable. These findings are in contrast to previous results showing that the late-infantile type of MLD is always associated with the complete absence of ARSA activity. The expression of the mutant ARSA protein may be influenced by particular features of oligodendrocytes, such that the level of mutant enzyme is lower in these cells than in others.

Topics: Alleles; Animals; Base Sequence; Cells, Cultured; Cerebroside-Sulfatase; Cricetinae; DNA Mutational Analysis; Fluorescent Antibody Technique; Frameshift Mutation; Genotype; Glycine; Humans; Infant; Leukodystrophy, Metachromatic; Male; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodendroglia; Phenotype; Point Mutation; Rhodamines; Serine; Sulfoglycosphingolipids

The lysosomal removal of the sulfate moiety from sulfatide requires the action of two proteins, arylsulfatase A and sphingolipid activator protein-1 (SAP-1). Recently, patients have been identified who have a variant form of metachromatic leukodystrophy which is characterized by mutations in the gene coding for SAP-1, which is also called "prosaposin." All of the mutations characterized in these patients result in (a) deficient mature SAP-1, as determined by immunoblotting after SDS-PAGE of tissue and cell extracts, and (b) decreased ability of cultured skin fibroblasts to metabolize endocytosed [14C]-sulfatide. We now report the insertion of the full-length prosaposin cDNA into the Moloney murine leukemia virus-derived retroviral vector, pLJ, and the infection of cultured skin fibroblasts from a newly diagnosed and molecularly characterized patient with SAP-1 deficiency. The cultured cells infected with the prosaposin cDNA construct now show both production of normal levels of mature SAP-1 and completely normal metabolism of endocytosed [14C]-sulfatide. These studies demonstrate that the virally transferred prosaposin cDNA is processed normally and is localized within lysosomes, where it is needed for interaction between sulfatide and arylsulfatase A. In addition, normal as well as mutant sequences can now be found by allele-specific oligonucleotide hybridization of PCR-amplified genomic DNA by using exonic sequences as primers.

Topics: Antisense Elements (Genetics); Base Sequence; Cells, Cultured; Cerebroside-Sulfatase; Child; Fibroblasts; Genetic Vectors; Glycoproteins; Humans; Kinetics; Leukodystrophy, Metachromatic; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides; Plasmids; Polymerase Chain Reaction; Protein Precursors; Retroviridae; Saposins; Skin; Sphingolipid Activator Proteins; Stearic Acids; Sulfoglycosphingolipids; Transfection

Sulfatide excretion in urine and arylsulfatase A (ASA) activity in leukocytes were determined in 10 homozygotes of metachromatic leukodystrophy (MLD), 7 obligate and 5 facultative MLD heterozygotes, 6 low ASA subjects (not related to MLD homozygotes), and in 9 controls. As compared to controls (sulfatides: 0-2 nmol/mg lipid; ASA: 101-287 nmol p-nitrocatechol/mg protein/hr), MLD homozygotes displayed highly increased sulfatide excretions (27-280 nmol) and low residual ASA activities (0-13 nmol). Of 12 MLD heterozygotes (ASA: 18-87 nmol) 10 showed increased sulfatides (3-24 nmol). All heterozygotes with ASA activity < 60 nmol (n = 8) had elevated sulfatide excretions (4-24 nmol). Thus, reduction of ASA activity below 40% of the mean value of controls seems to be the critical threshold for elevated sulfatide excretion in MLD heterozygotes. The low ASA subjects (ASA in the heterozygote range) excreted sulfatides in the control range, even those with ASA activities < 60 nmoles (n = 3; including a definite homozygote for ASA-pseudodeficiency; ASA:25 nmol). Statistical evaluation of sulfatide excretion and ASA activity in all subjects (n = 37) revealed a significant inverse relation (Spearman rank correlation; R = 0.8278, P < 0.001). The finding of elevated sulfatide excretion in certain MLD heterozygotes might point to increase of sulfatides also in the nervous system.

Topics: Adult; Cerebroside-Sulfatase; Female; Heterozygote; Homozygote; Humans; Infant; Leukocytes; Leukodystrophy, Metachromatic; Male; Sulfoglycosphingolipids

We report on a family with a sibship of three children for whom the diagnosis of "an unusual form of metachromatic leukodystrophy (MLD)" had been suggested earlier. The patients had choreiform movements and dystonic posturing accompanied by dysarthria since childhood. The availability of the polymerase chain reaction enabled us to show that the three siblings have a pseudodeficiency genotype (ASAp/ASAp). There was no abnormal sulphatiduria, and we propose that the neurological disease and low arylsulphatase A activity are unrelated to one another in this family. A diagnosis of MLD carries very serious implications, and we recommend that gene amplification by polymerase chain reaction and hybridization with allele-specific oligonucleotide probes should be used to corroborate the diagnosis, especially when there is no abnormal sulphatiduria and when metachromatic material cannot be demonstrated in a sural nerve biopsy.

Topics: Adolescent; Adult; Athetosis; Cerebroside-Sulfatase; Chorea; Diagnosis, Differential; Dysarthria; Female; Homozygote; Humans; Leukodystrophy, Metachromatic; Male; Polymerase Chain Reaction; Sulfoglycosphingolipids

A 7-year-old boy had clinical features of metachromatic leucodystrophy (MLD), however, an increased urinary sulphatide excretion was found in the presence of normal arylsulphatase A (and alpha-galactosidase A) activity. A rectal biopsy showed metachromatically staining storage macrophages as well as nonmetachromatic, but PAS-positive, submucosal neurons filled with membranous cytoplasmic bodies. These two types of storage material led to testing for a sphingolipid activator protein (SAP) deficiency. Loading tests with sulphatide and globotriaosylceramide showed deficient turnover of both sphingolipids in cultured fibroblasts. Using the Ouchterlony method, there was no reactivity between a described anti-SAP 1 antiserum and the patient's fibroblast extracts. This new case of SAP-1 deficient MLD was compared with the four cases of this variant known from the literature. Our results indicate that rectal biopsy morphology and lipid loading biochemistry should prove useful for the screening of SAP defects.

Topics: Biopsy; Cerebroside-Sulfatase; Child; Glycoproteins; Humans; Immunologic Techniques; Leukodystrophy, Metachromatic; Male; Rectum; Saposins; Sphingolipid Activator Proteins; Sulfoglycosphingolipids

We report an improved method for the radiolabelling of [1-14C 00acyl-sphingosine-galactose-3-sulfate (sulfatide), requiring preparation of lysosulfatide (sulfogalactosyl-sphingosine) by alkaline hydrolysis of sulfatide and reacylation of the sphingosine amino group with a [1-14C]stearoyl chloride. We found that the yield of labeled sulfatide could be considerably increased using stringent chromatographic conditions for the preparation of lysosulfatide and strict anhydrous conditions for the formation of the acylchloride and its coupling to lysosulfatide. Radioscanning was used at different steps to check the purity of the labeled compounds. Radioscanning was also used to determine the formation of cerebroside when measuring cerebroside sulfate sulfatase activity and sulfatide metabolism in intact fibroblasts in controls and patients with metachromatic leukodystrophy. It could demonstrate and measure with accuracy the cerebroside sulfate storage characteristic of the disease.

Topics: Carbon Radioisotopes; Cells, Cultured; Cerebroside-Sulfatase; Fibroblasts; Humans; Leukodystrophy, Metachromatic; Psychosine; Radionuclide Imaging; Sulfoglycosphingolipids

Topics: Age Factors; Bone Marrow Transplantation; Cerebroside-Sulfatase; Child; Female; Humans; Leukocytes; Leukodystrophy, Metachromatic; Sulfoglycosphingolipids

Topics: Cells, Cultured; Colorimetry; Fibroblasts; Humans; Leukodystrophy, Metachromatic; Sulfoglycosphingolipids; Tritium

Ultrastructure of cellular inclusions in the cerebral cortex and in renal tubule epithelial cells in a case of late infantile metachromatic leukodystrophy were investigated. Four patterns of shape were illustrated as multi-lamellated structure, zebra bodies, dense bodies and vacuoles with the two latter being more predominant in the renal tubule cells. Histochemical studies of the cellular deposits with toluidine blue and cresyl violet all revealed metachromatic in character. Significance of these findings and their pathogenesis were discussed.

Topics: Cerebral Cortex; Child, Preschool; Female; Histocytochemistry; Humans; Inclusion Bodies; Kidney Tubules; Leukodystrophy, Metachromatic; Sulfoglycosphingolipids

Two fluorescent derivatives of cerebroside sulfate ('sulfatide') have been synthesized and used as substrates for determining arylsulfatase A activity. These were 12-(1-pyrene)dodecanoyl cerebroside sulfate (P12-sulfatide) and 12(1-pyrenesulfonylamido)dodecanoyl cerebroside sulfate (PSA12-sulfatide). When incubated at pH 5.0 in the presence of 5 mM MnCl2 and 5.5 mM of taurodeoxycholate, either substrate was hydrolyzed by arylsulfatase A of human leukocytes. The rate of hydrolysis was proportional to the incubation time and concentration of enzyme; Michaelis-Menten type kinetics were observed with increasing concentrations of substrate. For determining the rate of hydrolysis, each of the two products (i.e., P12- and PSA12-cerebrosides) were separated from the bulk of respective unreacted sulfatide on small columns of DEAE-Sephadex A-25 and their fluorescence intensities read at 343-378 and 350-380 nm for the excitation and emission wavelengths for P12- and PSA12-cerebrosides, respectively. When extracts of skin fibroblasts derived from normal individuals and patients with Maroteaux-Lamy (lacking arylsulfatase B) or metachromatic leukodystrophy (lacking arylsulfatase A) were used as source of enzyme, P12-sulfatide was hydrolyzed by the former two but not by the latter cell extract. Several derivatives of cerebroside sulfate were also synthesized and found to inhibit the hydrolysis of pyrenesulfatide by leukocyte arylsulfatase A. The results demonstrate that these two pyrene containing sulfatides can be effectively used as specific substrates for the determination of arylsulfatase A activity in extract of cells and most probably also of tissues.

Topics: Animals; Cattle; Cerebroside-Sulfatase; Cerebrosides; Fluorescent Dyes; Humans; Hydrolysis; Kinetics; Leukocytes; Leukodystrophy, Metachromatic; Magnetic Resonance Spectroscopy; Mucopolysaccharidosis VI; Pyrenes; Spectrometry, Fluorescence; Spectrophotometry; Substrate Specificity; Sulfoglycosphingolipids

Metachromatic leukodystrophy (MLD) is a rare inherited neurodegenerative disease associated with a defect in the catabolism of sulphatide (galactocerebroside-sulphate) which accumulates in the nervous system. MLD can be diagnosed biochemically by demonstrating deficiency in the activity of the enzyme arylsulphatase A (ASA) and an excess of sulphatide in urine and tissues. Clinically adult MLD may present as a schizophrenic-like psychosis, which typically develops years before the onset of neurologial signs which are not inevitable. Urinary ASA was investigated in 99 chronic hospitalized psychiatric patients (including 77 schizophrenics). Thirteen showed reduced ASA activity. Of the nine who were available for further study, only one evinced reduced ASA activity in other tissues (for example, leukocytes and cultured fibroblasts). However, there was no evidence of sulphatidurea with impaired sulphatide hydrolysis in his intact cultured fibroblasts. Therefore, he tested negative for MLD. This biochemical profile is known as pseudosulphatase deficiency. The possible relationship of pseudosulphatase deficiency to schizophrenic-like conditions is discussed.

Topics: Cerebroside-Sulfatase; Diagnosis, Differential; Humans; Leukodystrophy, Metachromatic; Neurocognitive Disorders; Schizophrenia; Schizophrenic Psychology; Sulfoglycosphingolipids

In a child with enzymatically and histopathologically proven metachromatic leukodystrophy (MLD), the disease pursued a course typical of juvenile MLD characterized by neurological degeneration beginning at age 9 years and ending in death at age 18. A younger brother of the patient was found to have profound deficiency of arylsulfatase A in leukocytes and to excrete five- to 20-fold greater-than-normal amounts of sulfatide in the urine. He was completely free of symptoms attributable to MLD until age 16 when he developed acute cholecystitis caused by sulfatide accumulation in the gallbladder. Results of detailed neurological examination at age 21 years were normal; formal psychometric assessment showed a full-scale IQ of 105 (Wechsler). Studies on cultured skin fibroblasts from the brother showed defects in arylsulfatase A activity, measured with the use of synthetic and natural substrates, and in radiolabeled sulfatide turnover. Cellulose acetate gel electrophoresis of fibroblast extracts from the patient showed no detectable arylsulfatase A isozyme under conditions that clearly distinguished pseudo-arylsulfatase A deficiency from classical MLD. Biochemically, the patient was indistinguishable from patients with classical MLD; on the other hand, his clinical course is dramatically more benign than that of his sister who was affected with severe MLD.

Topics: Adolescent; Cerebroside-Sulfatase; Child; Electrophoresis, Cellulose Acetate; Female; Fibroblasts; Humans; Leukocytes; Leukodystrophy, Metachromatic; Male; Mutation; Neurologic Examination; Pedigree; Sulfoglycosphingolipids

This study aimed at increasing the efficiency and shortening the time required for administering cerebroside sulfate to cultured cells. For this purpose several modes of dispersion of a fluorescent derivative of cerebroside sulfate (sulfatide), in which the natural fatty acid has been replaced by pyrenedodecanoic acid (P12), were incubated with the cells. This fluorescent derivative of cerebroside sulfate (P12-CS) was introduced into the growth medium of the cells using the three following modes of dispersion: (1) P12-CS was dissolved in dimethylsulfoxide and added to the medium, (2) it was precomplexed with serum albumin or (3) incorporated into small, unilamellar vesicles (SUV) of phosphatidylcholine. With each of these respective modes of dispersion, the P12-CS was incubated for periods up to 48 h with cultured lymphoblasts or fibroblasts. Uptake by the cells could be determined by recording directly the cell-associated fluorescence, using a suspension of washed intact cells. The cell lipids were subsequently extracted with mixtures of chloroform/methanol and their fluorescence recorded. When related to the incubation time, uptake of P12-CS by the cells increased continuously using each of the above dispersions. The appearance of fluorescence at 475 nm ('excimer') and the ratio of this to the monomolecular fluorescence at 378 nm ('E/M') could be used as a measure for the presence of the internalized P12-CS in aggregated or fully dispersed states. These values (i.e., E/M), recorded on the suspensions of intact cells were rather high using the aqueous dispersions, intermediate values were observed using the SUV and rather low E/M values (0.5 or less) were observed using the preformed albumin-(P12-CS) complexes. Increasing the mole ratio of albumin to P12-CS (i.e., from 1:2 to 2:1 m/m), decreased the quantity of sulfatide which was taken up by the cells but also further decreased the E/M ratio, suggesting a fully dispersed state of the pyrene lipid within the cell. This indicated that, using an optimal albumin to P12-CS ratio of 1-2 (or its equivalent values in fetal calf serum) permitted an influx of single molecules of P12-CS into the cells. After 48 h, about 50% of the fluorescence of skin fibroblasts was found in metabolic degradation products of P12-CS. The parallel value for fibroblasts derived from a patient with metachromatic leukodystrophy was only about 5%. Appearance of the excimeric emission of a dispersion of P12-CS in water permitted estimat

Topics: Cell Line; Fibroblasts; Fluorescent Dyes; Humans; Kinetics; Leukodystrophy, Metachromatic; Liposomes; Lymphocytes; Micelles; Serum Albumin, Bovine; Spectrometry, Fluorescence; Sulfoglycosphingolipids

The glycosphingolipid pattern was examined in three cases of late infantile metachromatic leukodystrophy (MLD): one with a relatively short (2.5 years), one with a long (7.8 years), and one with a very long (13.2 years) survival time. All values were compared with those of age-matched normal controls. The cerebroside concentration was reduced to 25, 12, and 4%, respectively, in the MLD white matter, whereas the sulfatide concentration was increased up to 200% of the control value. The yield of myelin was reduced to less than 15% in the early case and to less than 3 and 1%, respectively, in the two later cases. There was no sign of increased sulfatide proportion in the myelin. The ganglioside pattern was normal in cerebral gray matter, but in the white matter, contents of gangliosides of the lacto series were significantly increased, in particular, the ganglioside suggested by us as being characteristic of reactive astrocytosis. For the first time, lysosulfatide was identified in MLD and normal human brains by mass spectrometry and radioimmunoaffinity TLC using specific monoclonal antibody. Its quantity was found to be similar in normal and MLD brains. These findings support our postulation that the lysoglycosphingolipids are synthesized de novo from sphingosine and that they do not play a key role in pathogenetic mechanisms.

Topics: Adolescent; Brain; Cerebrosides; Child; Child, Preschool; Female; Glycolipids; Humans; Leukodystrophy, Metachromatic; Lipid Metabolism; Male; Myelin Sheath; Psychosine; Reference Values; Sphingosine; Sulfoglycosphingolipids; Tissue Distribution

Topics: Adolescent; Cerebroside-Sulfatase; Child, Preschool; Gene Expression Regulation; Genes, Recessive; Humans; Infant; Leukodystrophy, Metachromatic; Sulfoglycosphingolipids

A fatal case of multiple sulfatase deficiency in a 10-year-old girl is reported. In this rare disease, which is inherited as an autosomal recessive, features of metachromatic leukodystrophy and of mucopolysaccharidoses occur together. The white matter suffers progressive destruction with sulfatides accumulating in macrophages; these stain metachromatically as golden brown granules with acetic acid thionin stain. Cortical and subcortical neurons are distended by lipids and mucopolysaccharides, and fibrous thickening of the leptomeninges leads to severe obstructive hydrocephalus. This entity can be regarded as a bridge between leukodystrophies and neuronal storage diseases both conceptually and in its morphological manifestations.

Topics: Brain; Brain Chemistry; Child; Female; Glycosaminoglycans; Humans; Leukodystrophy, Metachromatic; Lipids; Macrophages; Microscopy, Electron; Mucopolysaccharidoses; Spinal Cord; Sulfatases; Sulfoglycosphingolipids

Topics: Brain; Humans; Kidney; Leukodystrophy, Metachromatic; Lysosomes; Sulfatases; Sulfoglycosphingolipids

A 4-year old boy died of diffuse disseminated sclerosis (DDS) of the brain and was found to have also pseudoarylsulfatase A deficiency (PASAD) with about 20% residual arylsulfatase A (ASA) and cerebroside sulfatase (CS) activity. The reexamination of lipids did not show any sulfatide accumulation in the patient's organ extracts. Although the residual CS activity in the patient's extracts was clearly demonstrable only after partial purification, it was concluded that this activity protects organ tissues from sulfatide accumulation in PASAD, since in sulfatide lipidosis (metachromatic leukodystrophy, MLD) no residual CS activity was detectable. The study of residual ASA activity in the patient's fibroblasts by gel electrofocusing resulted in an almost normal enzyme microheterogeneity. However, the detailed study of the brain galactolipids in the patient revealed an elevated ratio of sulfatide/galactocerebroside content, despite the decrease of both lipids. In tissues of other patients with severe demyelinating diseases different from DDS and MLD, this galactolipid ratio was also found to be increased, especially in three patients with adrenoleukodystrophy. A general mechanism of this anomaly in severe demyelination is considered.

Topics: Adolescent; Adrenoleukodystrophy; Adult; Brain; Cerebroside-Sulfatase; Child; Child, Preschool; Diffuse Cerebral Sclerosis of Schilder; Female; Galactolipids; Galactosylceramides; Glycolipids; Humans; Infant; Leukodystrophy, Metachromatic; Male; Middle Aged; Multiple Sclerosis; Sulfoglycosphingolipids

Arylsulfatase A (ASA) deficiency is the cause of early and late onset metachromatic leukodystrophy (MLD). Low ASA levels are detected in some healthy individuals who are pseudodeficient (PD). PD individuals can be distinguished, because PD fibroblasts hydrolyze 14C-sulfatide at similar rates to normal fibroblasts. This has also been demonstrated in amniocytes and chorionic villi (CV). The genetic basis for PD is not clearly understood and is most likely heterogeneous with respect to allelic mutations of the ASA gene. It is hypothesized that the PD phenotype can either be due to PD/PD or PD/MLD genotypes, only the latter representing a potential risk to offspring. We report an unusual family where two siblings, both carriers of the classic late infantile MLD allele, are married to unrelated PD individuals. One couple has two PD offspring; their "at risk" status, due to the lack of an affected offspring is in question. The other couple terminated a fetus determined to be affected with a "MLD variant", most likely a compound heterozygote. Cautions prenatal counseling of PD families is essential. The population frequency of the PD phenotype is unknown.

Topics: Cerebroside-Sulfatase; Female; Genetic Counseling; Heterozygote; Humans; Leukodystrophy, Metachromatic; Male; Pedigree; Pregnancy; Prenatal Diagnosis; Risk; Sulfoglycosphingolipids

A fluorescent derivative of cerebroside sulfate (12-(1-pyrene)dodecanoyl-sphingosylgalactosyl-0-3-sulfate (P12-sulfatide) has been synthesized as a potential substrate for the determination of cerebroside sulfatidase (or arylsulfatase A) activity. It was administered into cultured human skin fibroblasts and thereby utilized for the diagnosis of arylsulfatase A deficiency. Cultured skin fibroblasts from normal individuals and healthy persons suffering from a pseudoarylsulfatase A deficiency (PD) degraded the P12-sulfatide, while in cells derived from a metachromatic leukodystrophy (MLD) patient it remained essentially intact. This contrasts with in vitro determinations of enzymatic activity, where the MLD or PD-derived arylsulfatase A exhibit similar deficiency, in spite of a profoundly different clinical course. Administration of the fluorescent sulfatide into the intact cells permitted a sensitive and rapid diagnosis of MLD and its distinction from the PD-phenomenon. This might be of particular importance for cases in which a rapid diagnosis is required and for prenatal diagnosis of fetuses from families afflicted with both MLD and pseudo-deficiency mutant genes.

Topics: Cell Line; Cerebroside-Sulfatase; Female; Fluorescent Dyes; Humans; Leukodystrophy, Metachromatic; Pregnancy; Prenatal Diagnosis; Sulfoglycosphingolipids

A simple procedure was developed to assay the ability of arylsulfatase A in extracts of cultured skin fibroblasts to degrade the natural substrate, sulfatide, in the presence of the physiological activator protein but without detergents. Inhibitory substances were removed by dialysis and by batch-wise ion-exchange chromatography. The enzyme recoveries during purification were monitored with a newly developed method that employs the chromogenic substrate 4-nitrocatecholsulfate at an incubation temperature of 4 degrees C. The residual sulfatidase activities determined with this procedure in fibroblasts from patients with various forms of MLD correlated well with the severity of the disease.

Topics: Cells, Cultured; Cerebroside-Sulfatase; Chromatography, DEAE-Cellulose; Enzyme Activation; Fibroblasts; Humans; Hydrogen-Ion Concentration; Hydrolysis; Leukodystrophy, Metachromatic; Micelles; Sulfoglycosphingolipids

A patient with a new variant of multiple sulphatase deficiency (MSDv) is reported. Unlike the usual type, onset was late and progress was slow. The phenotypic changes were those usually seen in multiple sulphatase deficiency but much milder. Cytoplasmic accumulations were found in skin fibroblasts, and urinary mucopolysaccharides and sulphatides were high. Arylsulphatases A, B and C (ASA, B and C), heparan N-sulphatase sulphoiduronate sulphatase, and N-acetylgalactosamine 6-sulphatase all had low activity in lymphocytes and cultured skin fibroblasts. Complementation for ASA activity was found in hybrids between MSDv and metachromatic leukodystrophy (MLD) as well as between multiple sulphatase deficiency (MSD) and MLD. Complementation for ASC activity was also seen in hybrids between MSDv and X-linked ichthyosis (XLI), and between MSD and XLI. However, neither ASA nor ASC activity increased in hybrid cells of MSDv and MSD. These results suggested that the mutations of MSDv and of MSD were allelic, although of different phenotypes.

Topics: Adult; Alleles; Arylsulfatases; Female; Genetic Complementation Test; Glycosaminoglycans; Humans; Hybridization, Genetic; Leukodystrophy, Metachromatic; Metabolism, Inborn Errors; Mutation; Sulfatases; Sulfoglycosphingolipids

Metachromatic leukodystrophy (MLD) is a disease caused by a deficiency of the enzyme sulfatide sulfatase, also known as arylsulfatase A (ASA). We compared the activity of this enzyme in adult psychiatric patients and normal volunteers using nitrocatechol sulfate (ASA-NCS) and cerebroside sulfate (ASA-CS) as substrates. Our results showed that ASA-NCS activity in urine and leukocytes was significantly lower in psychiatric than in normal individuals, but that there were no differences between these two groups in the sulfatide excretion in urine or the ASA-CS activity in leukocytes. There was no correlation between enzyme activity in urine and in leukocytes, indicating that activity in urine does not truly reflect the levels of the enzyme in tissues. The correlation between ASA-NCS and ASA-CS activity in leukocytes was poor (0.51 for psychiatric patients and 0.59 for normals), suggesting that for a valid measure of the enzyme activity the assays should be carried out with CS as substrate. Results of our study also indicate that in 39 of the 145 psychiatric patients studied, the ASA-CS activity in leukocyte was less than 4 nmoles/mg protein/hr, which is below 50% of the normal means, whereas only one of the 30 normal subjects had a value this low. The presence of low levels of ASA-CS activity in a significantly large number of adult patients with varying psychiatric manifestations suggests that such patients may be asymptomatic carriers of the sulfatidase defect (heterozygotes for MLD), and that behavioral and functional disturbances in these patients may at least in part be related to sulfatidase deficiency. The significance of the ASA-NCS abnormality (reduction) in psychiatric patients is unclear.

Topics: Cerebroside-Sulfatase; Female; Humans; Huntington Disease; Intellectual Disability; Leukocytes; Leukodystrophy, Metachromatic; Male; Mental Disorders; Neurocognitive Disorders; Schizophrenia; Sulfatases; Sulfoglycosphingolipids

A woman aged 21 with a variant form of metachromatic leucodystrophy (MLD) combined with another form of leucodystrophy is described. The clinical symptoms were retinitis pigmentosa and progressive neurological deficits such as mental retardation, dystonia, pyramidal tract involvement and peripheral neuropathy. The biochemical findings were marked deficiency of arylsulfatase-A and cerebroside-sulfatase in cultured fibroblasts and excretion of sulfatides in the urine. Sulfatide-loading of cultured fibroblasts showed almost normal uptake and degradation of sulfatides. The patient's sister suffers from a clinically similar neurological disease, but normal activity of arylsulfatase-A was found in her leucocytes. A severe oral-facial dystonia in the patient was successfully controlled by l-dopa.

Topics: Adult; Central Nervous System Diseases; Cerebroside-Sulfatase; Female; Fibroblasts; Humans; Intellectual Disability; Leukocytes; Leukodystrophy, Metachromatic; Nerve Degeneration; Neuromuscular Diseases; Retinitis Pigmentosa; Sulfoglycosphingolipids

Metachromatic leukodystrophy (MLD) presents as six separate variant forms, four allelic and two non-allelic. It is diagnosed in the laboratory by a decrease in the fibroblast or leukocyte arylsulfatase A activity, generally against an artificial substrate. Since residual enzyme activity is not always an indicator of presence or absence of disease, it may be helpful to supplement this information with that of the presence or absence of sulfatide storage in the body. We have improved the HPLC analysis of sulfatide by the use of a sulfated internal standard, sulfatoxymonoalkylmonoacylgalactosylglycerol. Normal urines contain approximately 0 to 0.2 nmol sulfatide/mg creatinine, whereas MLD urines may contain 5 to 7.5 nmol/mg. There is no increase in plasma sulfatide compared to controls in the age group of MLD patients which we studied (up to 4 years).

Topics: Cerebroside-Sulfatase; Chromatography, High Pressure Liquid; Glycolipids; Glycosphingolipids; Humans; Leukocytes; Leukodystrophy, Metachromatic; Sulfoglycosphingolipids

The urinary sulfatides in metachromatic leukodystrophy (MLD) were analyzed by gas chromatography-mass spectrometry. Fatty acids and long chain bases were obtained after methanolysis. C22:0 and C22h:0 were major components of the fatty acids distributed in the urinary sulfatides in MLD while they were only minor components of the fatty acids in the brain sulfatides in a control subject. These results were in accordance with the report of Philippart et al. It was suggested that the urinary sulfatides originated not in the brain but in other organs. The mass spectra of the trimethylsilyl derivatives of the hydroxy fatty acid methyl esters always showed peaks at m/z (M-15-28)+ and (M-59)+, indicating that the hydroxy group was on carbon 2. Two kinds of long chain base were identified: C18-sphingosine and 3-O-methyl-C18-sphingosine. The latter compound may be a by-product formed on methanolysis.

Topics: Brain Chemistry; Chemical Phenomena; Chemistry; Child, Preschool; Chromatography, Gas; Fatty Acids; Humans; Leukodystrophy, Metachromatic; Male; Mass Spectrometry; Sphingosine; Sulfoglycosphingolipids; Vitamin A

We describe two boys, presenting by 1 year of age, with developmental delay from birth, mildly coarse facial features, and hepatomegaly. These clinical features were most suggestive of a mucopolysaccharidosis, particularly MPS II. Biochemical studies, including sulfate incorporation in fibroblasts and lysosomal enzyme analyses in fibroblasts, leukocytes, and serum, showed abnormalities in both sulfatide and mucopolysaccharide metabolism and led to the diagnosis of multiple sulfatase deficiency. With time, both patients developed an ichthyotic rash and profound intellectual deterioration. We conclude that findings in the first year of life in some patients with MSD may closely resemble those in patients with a MPS disorder rather than the late infantile form of metachromatic leukodystrophy, as is classically described. Thus, MSD should be considered in the young patient suspected of having a MPS disorder.

Topics: Cerebroside-Sulfatase; Child, Preschool; Chondro-4-Sulfatase; Diagnosis, Differential; Fibroblasts; Glycosaminoglycans; Hexosaminidases; Humans; Infant; Leukocytes; Leukodystrophy, Metachromatic; Male; Mucopolysaccharidoses; Skin; Sulfatases; Sulfoglycosphingolipids; Uronic Acids

A 3 1/2-year-old slightly retarded boy with marked deficiency of arylsulfatase A (ASA) activity in leucocytes and fibroblasts and almost no cerebroside sulfatase (CS) activity in fibroblasts was tested with the sulfatide-loading test. On this test his fibroblasts showed impaired degradation. A pathological excretion of sulfatides was seen in his urine. Nerve conduction velocity, visual evoked potential, auditory brain stem evoked response, and somatosensory evoked potential were all normal. His father and older brother had similarly low levels of ASA in leucocytes and fibroblasts and 1.7-2% residual CS activity in fibroblasts. Although both were clinically normal, their fibroblasts accumulated increased amounts of sulfatides when challenged in the sulfatide-loading test. In this family, this test thus will be of no value in prenatal diagnosis to discriminate among low ASA fetuses with pseudoarylsulfatase A deficiency and fetuses with this unusual ASA deficiency variant.

Topics: Adult; Cells, Cultured; Cerebroside-Sulfatase; Child, Preschool; Female; Fibroblasts; Genetic Variation; Humans; Leukocytes; Leukodystrophy, Metachromatic; Male; Pregnancy; Prenatal Diagnosis; Sulfatases; Sulfoglycosphingolipids; Sulfur Radioisotopes

Among the genetic leukodystrophies known to occur in man, the fundamental genetic defects have been clarified in two disorders, metachromatic leukodystrophy and globoid cell leukodystrophy (Krabbe's disease). Nevertheless, the biochemical sequences with which the underlying genetic defects lead to the clinical and pathological features of the diseases and to disruption of the normal brain function are not well understood. Comparison of the two classical leukodystrophies provides us an opportunity to consider the biochemical pathogenesis of these disorders, some common between them and others apparently unique. That both metachromatic leukodystrophy and globoid cell leukodystrophy manifest themselves almost exclusively as diseases of the myelin sheath is relatively easy to understand because both are caused by genetic abnormalities in the metabolism of the characteristic constituents of myelin, sulfatide and galactosylceramide. The presence of the abnormal and characteristic globoid cells in the white matter of Krabbe's disease patients appears to be due to a unique property of galactosylceramide in that, when present in free form in the brain, it elicits infiltration of macrophages which transform themselves to globoid-like cells. No other lipids, including sulfatide, are known to induce similar tissue reactions. The most conspicuous difference between the two diseases is the presence or absence of abnormal accumulation of the substrates, the degradation of which is genetically blocked in the respective diseases. In metachromatic leukodystrophy, sulfatide accumulates to abnormally high levels, as logically expected for a "lysosomal storage disease". In Krabbe's disease, on the other hand, galactosylceramide is always much less than normal despite the genetic block in its catabolic pathway. This paradoxical finding can be explained by the "psychosine hypothesis".(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Brain; Cerebroside-Sulfatase; Child; Diffuse Cerebral Sclerosis of Schilder; Galactosylceramidase; Galactosylceramides; Humans; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Myelin Sheath; Oligodendroglia; Psychosine; Sulfoglycosphingolipids

Metachromatic leukodystrophy is a recessively inherited disease of children and adults. The basic disorder is a failure of the catabolism of sulfatide, the sulfate ester of galactose cerebroside. This lipid is a component of the myelin membrane and is probably a component of neuronal membranes as well. The various forms of clinical presentation, the aids to diagnosis, the genetic variations of arylsulfatase A, the enzyme involved in sulfatide catabolism, and possible approaches to therapy are presented.

Topics: Adolescent; Adult; Cerebroside-Sulfatase; Child; Child, Preschool; Diagnosis, Differential; Fibroblasts; Genetic Carrier Screening; Humans; Infant; Leukocytes; Leukodystrophy, Metachromatic; Middle Aged; Neural Conduction; Sulfoglycosphingolipids

Leukodystrophies are disorders affecting primarily oligodendroglial cells or myelin. Another necessary criteria for defining a leukodystrophy is that the disorder has to be of endogenous origin with a pattern compatible with genetic transfer of a metabolic defect. The clinical criterion of a steadily progressive deterioration of function must also be included. Much of the material presented during the conference related to three leukodystrophies from which subclasses with a relatively uniform clinical presentation can be distinguished and about which a considerable body of consistent biochemical information is available. These disorders are metachromatic leukodystrophy (MLD), globoid leukodystrophy (GLD, also referred to as Krabbe's disease) and adrenoleukodystrophy (ALD). Discussion was focused to the pathophysiology of the more prevalent "classical" subclass of each of these disorders; discussion of clinical variants was in the context of what we could learn about the more prevalent form. Finally, because of time restraints, most of the discussion on the final day was centered around MLD. This clinical disorder was used as a starting point for discussion of questions both specific to MLD as well as those questions common to all leukodystrophies. The discussion of GLD and ALD was more restricted and only points relevant to those specific leukodystrophies were discussed. Information from the presentation not dealing directly with these human disorders is also summarized.

Topics: Adrenoleukodystrophy; Animals; Child; Diffuse Cerebral Sclerosis of Schilder; DNA, Recombinant; Fatty Acids; Galactosylceramidase; Genes, Recessive; Humans; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Mice; Mice, Neurologic Mutants; Mutation; Myelin Proteins; Psychosine; Sulfoglycosphingolipids

A child with a diagnosis of late-infantile metachromatic leukodystrophy (MLD), and a normal father with low arylsulfatase A (ASA) activity in leucocytes and cultured fibroblasts is described. The child had a pathologically increased amount of sulfatides in the urine, whereas no sulfatides could be found in the father's urine. Sulfatide-loading of the child's cultured fibroblasts showed an accumulation of sulfatides, whereas the fibroblasts from the father had a marginally decreased sulfatide turnover. It is thus possible to discriminate between these two forms of low ASA activity in this family, and to ensure a correct diagnosis should the amniotic fluid cells show a low ASA activity in future pregnancies.

Topics: Cerebroside-Sulfatase; Child, Preschool; Female; Fibroblasts; Humans; Leukocytes; Leukodystrophy, Metachromatic; Male; Sulfatases; Sulfoglycosphingolipids

The intact fibroblast cerebroside sulfate loading test is useful because sulfatide hydrolysis can be demonstrated in late onset MLD cell types with 1% or less of normal arylsulfatase A. In such cells, hydrolysis of sulfatide was inhibited when the loading test was carried out in growth media containing the organic ampholyte Hepes. Since Hepes did not affect uptake of sulfatide nor intracellular levels of arylsulfatase A, it was concluded that Hepes inhibited sulfatide hydrolysis by increasing lysosomal pH. The cerebroside sulfate loading test in the presence of Hepes should be useful as a probe for arylsulfatase A dysfunction in atypical MLD fibroblasts.

Topics: Animals; Cell Line; Culture Media; HEPES; Humans; Leukodystrophy, Metachromatic; Piperazines; Sulfoglycosphingolipids

In one 21-week-old fetus with prenatally diagnosed metachromatic leukodystrophy, galactolipid contents were determined in the forebrain cortex, cerebellum, brainstem, spinal cord, and kidney and compared to an appropriate control. Spinal cord and kidney showed the highest sulfatide accumulation as a consequence of deficient cerebroside sulfatase activity. No increase, but a measurable amount of sulfatide, was detected in the forebrain. The prenatal neural sulfatides contained a high proportion of the hydroxy fatty acid component. The galactosyl ceramides were not reduced in the early stage of the demyelinating disease.

Topics: Central Nervous System; Chromatography, Thin Layer; Female; Galactolipids; Glycolipids; Humans; Leukodystrophy, Metachromatic; Pregnancy; Pregnancy Trimester, Second; Sulfoglycosphingolipids

Late infantile metachromatic leukodystrophy (MLD) was successfully diagnosed in utero by demonstrating the absence of arylsulfatase-A in amniotic fluid using diethylaminoethyl-Sepharose column chromatography. Diagnosis by amniotic fluid using an ion-exchange column is more rapid and reproducible as compared with those reported previously. The diagnosis was confirmed by the absence of arylsulfatase-A in fetal brain, liver, and kidney tissues as well as by the marked accumulation of sulfatide in kidney. The kidney is the most appropriate organ for the demonstration of sulfatide accumulation in fetal tissues in MLD.

Topics: Amniotic Fluid; Brain; Cerebroside-Sulfatase; Child, Preschool; Chondro-4-Sulfatase; Chromatography; Humans; Kidney; Leukodystrophy, Metachromatic; Lipids; Liver; Male; Prenatal Diagnosis; Sulfoglycosphingolipids

Biochemical analyses were performed on cerebral biopsies from three siblings with metachromatic leukodystrophy, and from autopsy tissue obtained 6 months later. The lipids of all gray matter samples were relatively normal, with the exception of an elevated sulfatide: cerebroside ratio. The white matter samples were grossly abnormal and showed a progression in severity of biochemical abnormalities with duration of the disease. Sulfatides were 4-8-fold higher than normal, and the ratio of cerebrosides to sulfatides ranged from 0.13 to 0.26, compared to the normal value of about 4.0. Myelin was isolated in very low yield, but had normal morphology. As others have reported, it had the same chemical defect as whole white matter; sulfatides were 7-8 times higher than normal and cerebrosides were reduced by more than half. The fatty acid compositions of the myelin sphingolipids were found to be of much longer average chain length than those in affected white matter. In myelin, the unsubstituted fatty acids of cerebrosides and sphingomyelin had a higher percentage of short chains than found in normal myelin, but were less abnormal than those in whole white matter, whereas the unsubstituted and alpha-hydroxy acids of sulfatides and the alpha-hydroxy acids of cerebrosides were not deficient in long chains. These data indicate that there are at least two compartments of sphingolipids in MLD white matter, each having different average chain lengths.

Topics: Brain Chemistry; Cerebral Cortex; Cerebrosides; Chemical Phenomena; Chemistry; Child; Fatty Acids; Female; Humans; Leukodystrophy, Metachromatic; Male; Myelin Sheath; Phospholipids; Sphingolipids; Sulfoglycosphingolipids

Topics: Cells, Cultured; Cerebroside-Sulfatase; Child; Child, Preschool; Chromatography, High Pressure Liquid; Creatinine; Evoked Potentials, Auditory; Female; Fibroblasts; Humans; Infant; Leukodystrophy, Metachromatic; Male; Sulfoglycosphingolipids

Topics: Adolescent; Adult; Cerebroside-Sulfatase; Female; Humans; Leukocytes; Leukodystrophy, Metachromatic; Male; Middle Aged; Sulfatases; Sulfoglycosphingolipids

Continuing the previously published clinical development of a case of adult metachromatic leukodystrophy (MLD), we now describe the terminal phase and death (at 46 years of age) of our patient. The final phase was characterized clinically by progression of generalized peripheral neuropathy, advanced extrapyramidal and pyramidal tract symptomatology, dementia and brainstem dysfunction. First biochemical results show a moderate relative increase (3- to 5-fold) of sulfatides in the frontal lobe white matter but not in the cortex. The analysis of fatty acids in total lipid extract shows a decrease of long-chained fatty acids in favor of short-chained fatty acids, this change is more pronounced in white matter in the cortex. The clinical course and biochemical results are discussed in relation to previous cases analyzed by us. Epidemiological aspects especially emphasize routine serach for MLD amongst patients with neuropsychiatric symptomatology showing unusual psychoses, presenile dementias or unspecific disturbance of motor coordination possibly with electroneurographic evidence of peripheral neuropathy.

Topics: Age Factors; Arylsulfatases; Brain Chemistry; Fatty Acids; Female; Genetic Carrier Screening; Humans; Leukodystrophy, Metachromatic; Lipids; Middle Aged; Sulfoglycosphingolipids

A case of metachromatic leucodystrophy in a three years old female patient is reported. Histopathological examination revealed metachromatic inclusions within the cytoplasms of the neurons, which led the authors to comment on the physiopathogenesis of this disease, grouping it in category of the lipidoses.

Topics: Brain; Child, Preschool; Female; Humans; Leukodystrophy, Metachromatic; Neurons; Sulfoglycosphingolipids

A series of five living patients with juvenile metachromatic leukodystrophy (MLD), ten first-degree relatives, and a number of controls were subjected to biochemical investigations including quantitative determination of arylsulphatase A (ASA) and B (ASB) activities in peripheral leukocytes and polyacrylamide disc gel elctrophoresis of arylsulphatases. Five relatives were family members of four previously deceased patients with juvenile MLD. The mean ASA activity of the patients was 1.3 nmol of p-nitrocatechol sulphate hydrolysed in 30 min per mg protein. It was 84 nmol in the relatives, 129 nmol in other neurological patients and 136 nmol in normal controls. The corresponding ASB activity was 38 nmol in the patients, 49 nmol in the relatives, and 99 nmol in normal controls. An extremely low ASB activity, 3.4 nmol, was found in one relative. No ASA band could be visualised in the enzyme electrophoretic patterns of the patients' leukocytes but the bands representing ASB appeared normal. Seven relatives showed ASA bands weaker than normal, and the relative with low ASB activity exhibited very weak ASB band. The low ASB activity in the patients and heterozygotes may be a characteristic feature of the slowly progressive juvenile type MLD diagnosed in the present series.

Topics: Adult; Age Factors; Cerebroside-Sulfatase; Child; Chondro-4-Sulfatase; Electrophoresis, Polyacrylamide Gel; Female; Humans; Leukocytes; Leukodystrophy, Metachromatic; Male; Sulfatases; Sulfoglycosphingolipids

1. Follow-up studies of two siblings with mental retardation, progressive paraplegia and dementia were reported. 2. The brain and visceral organs of a patient (elder brother) who died recently were investigated histopathologically, electronmicroscopically and neurochemically. a. Moderate, diffuse demyelination occurred throughout the white matter of the central nervous system. b. Two abnormal materials were deposited in the white matter: one showed metachromasia containing sulfatide and another had staining characteristics of acid mucopolysaccharide histochemically. Electronmicroscopically, the former was a conglomerate of electron-dense materials of various degrees and the latter had a membrane-limited granular structure. The myocardium contained the same mucopolysaccharide material as that in the brain. c. Slight increase of sulfatide was found in the cerebral white matter. Arylsulfatase A activities were preserved in the brain as well as in the liver. Contents of hexosamine and uronic acid in the white matter were about three or five times as much as that of the controls. Electrophoresis on cellulose acetate membrane showed that acid glucopeptide was the main component of the mucopolysaccharide extracted from the brain.

Topics: Adult; Brain Chemistry; Cerebral Cortex; Electrophoresis, Cellulose Acetate; Female; Glycosaminoglycans; Hexosamines; Histocytochemistry; Humans; Leukodystrophy, Metachromatic; Male; Medulla Oblongata; Myocardium; Sulfoglycosphingolipids; Uronic Acids

Metachromatic leucodystrophies (MLD) comprise a small group of heredodegenerative disorders of the nervous system. Deficiency of sulfatide-sulfatase or arylsulfatase A is the common defect in all forms of MLD leading to lysosomal sulfatide storage in the nervous tissue and in the kidney. On the basis of animal experiments, experiments with cultured fibroblasts of the patients as well as ultrastructural studies in a case of prenatal MLD, the following pathomechanism is proposed: 1. Lysosomal degradation of a large portion newly synthetised sulfatide normally regulating the net synthesis and incorporation of sulfatide into myelin, causes early accumulation of sulfatide in lysosomes of myelinating cells and in neurons in the genetic deficiency of arylsulfatase A. 2. Early accumulation of sulfatide does not lead to disturbance in myelination. Demyelination occurs possibly by storage of a cytotoxic compound, psychosin sulfate, also a substrate for the missing enzyme. Prevention of MLD is possible by prenatal diagnosis of arylsulfatase A deficiency in cultured amniotic cells. Enzyme substitution of the missing arylsulfatase A is possible by exogenous uptake of the enzyme in cultured fibroblasts. Thereby the defect of sulfatide degradation can be corrected. Although principles of enzyme substitution have been demonstrated, the problems of treating patients with MLD with arylsulfatase A infusions have yet to be overcome.

Topics: Animals; Cells, Cultured; Cerebroside-Sulfatase; Humans; Kidney; Leukodystrophy, Metachromatic; Lysosomes; Mice; Myelin Sheath; Neurons; Oligodendroglia; Prenatal Diagnosis; Psychosine; Schwann Cells; Sulfoglycosphingolipids

Topics: Cerebroside-Sulfatase; Fibroblasts; Humans; Infant; Leukocytes; Leukodystrophy, Metachromatic; Male; Sulfoglycosphingolipids; Sural Nerve

Topics: Aged; Animals; Brain; Chromatography, Gas; Female; Fishes; Glycerides; Glycolipids; Humans; Leukodystrophy, Metachromatic; Male; Mass Spectrometry; Rats; Sulfates; Sulfoglycosphingolipids

Topics: Heterozygote; Humans; Leukodystrophy, Metachromatic; Prenatal Diagnosis; Sulfoglycosphingolipids

Topics: Adult; Brain Chemistry; Humans; Leukodystrophy, Metachromatic; Lipids; Male; Sulfoglycosphingolipids

In a 13-year-old neurologically healthy boy from a family with adult-onset of metachromatic leukodystrophy (MLD) showing arylsulfatase A-deficiency in the adult, sural nerve biopsy probably was performed 2-3 decades before clinical manifestation of the disease could be expected. Ultrastructurally 4 basic types of inclusion bodies in Schwann cells could be demonstrated (pleo-morphic "zebra body"-like inclusions, double-lamellated inclusions, "tuff-stone"-like inclusions, granular osmiophilic inclusions). Additionally, endoplasmatic reticulum, mitochondria and lysosomes showed marked alterations. Advanced damage of myelin was only rarely seen, but initial segmental demyelination was a common finding. These early pathological changes in chronic MLD are thought to represent a subcellular metabolic insufficiency of Schwann cells in this disease.

Topics: Adolescent; Cytoplasmic Granules; Endoplasmic Reticulum; Glycogen; Humans; Inclusion Bodies; Leukodystrophy, Metachromatic; Lipofuscin; Male; Microscopy, Electron; Mitochondria; Myelin Sheath; Peripheral Nerves; Schwann Cells; Sulfoglycosphingolipids

An autopsy case of adult metachromatic leukodystrophy (MLD) manifested clinically as schizophrenic psychosis is reported. A 50-year-old man developed progressive mental changes 10 years before his death, and later manifested a schizophrenic syndrome without neurologic deficits or EEG changes. After his death from uremia neuropathology disclosed MLD with demyelination accentuated in the frontal lobes and abundant metachromatic deposits in the preserved areas of cerebral white matter. Neurochemical examination of the demyelinated frontal area showed reduced concentration of cerebrosides and sulfatides, decreased amounts of total lipids in the tissue, and an increase of sulfatides, and particularly of their cerebron fractions in lipid extract. The problems of adult forms of MLD with prolonged course are discussed with special reference to cases showing mainly psychiatric syndromes.

Topics: Atrophy; Brain; Cerebrosides; Diagnostic Errors; Electroencephalography; Frontal Lobe; Humans; Hydrocephalus; Leukodystrophy, Metachromatic; Male; Middle Aged; Schizophrenia; Sulfoglycosphingolipids

Sural nerve biopsies taken from a pair of twins with metachromatic leukodystrophy were investigated by electron microscopy. The morphological findings showed distinct alterations. In the first twin the particularity was a large number of granules containing sulfatides. In the second twin, however, the main finding was a damage of myelin sheaths evidently caused by an excessive and morphologically as well as chemically defective myelin formation. According to the grading given by Ulrich the first case corresponds to the stadium one, in which a storage of sulfatides dominates, whereas case two showing a severe damage of myelin sheaths is characteristic of the second stadium of metachromatic leukodystrophy.

Topics: Biopsy; Diseases in Twins; Humans; Infant; Leukodystrophy, Metachromatic; Male; Microscopy, Electron; Myelin Sheath; Spinal Nerves; Sulfoglycosphingolipids; Sural Nerve

Topics: Cerebroside-Sulfatase; Child, Preschool; Heterozygote; Hexosaminidases; Homozygote; Humans; Leukodystrophy, Metachromatic; Sulfatases; Sulfoglycosphingolipids

Topics: Brain; Brain Chemistry; Ceramides; Humans; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Lysosomes; Sulfoglycosphingolipids

Polypoid masses metachromatic sulfatides have been found at autopsy in the gallbladder of patients with metachromatic leucodystrophy. In a 10-year-old girl with this disease oral cholecystrography demonstrated a filling defect, which was felt to represent a polyp. In the proper clinical setting, sulfatide cholecystosis should be considered in the differential diagnosis of polypoid lesions of the gallbladder.

Topics: Autopsy; Child; Female; Gallbladder Neoplasms; Humans; Leukodystrophy, Metachromatic; Polyps; Radiography; Sulfoglycosphingolipids

Alcian blue combines with purified sulphatide in 1.OM magnesium chloride. In tissue sections from patients with metachromatic leukodystrophy, sulphatide is stained by Alcian blue in 0.8 M magnesium chloride, and the staining can be abolished by prior treatment with chloroform and methanol. The simplicity of the technique, its specificity and ease of interpretation recommend Alcian blue staining at high salt concentrations as a routine method in the diagnosis of metachromatic leukodystrophy.

Topics: Alcian Blue; Brain Chemistry; Chlorides; Humans; Indoles; Kidney; Leukodystrophy, Metachromatic; Liver; Magnesium; Staining and Labeling; Sulfoglycosphingolipids

The fine structural characteristics of storage products in peripheral nerve, kidney and cerebral white matter, from a case of adult metachromatic leukodystrophy are described. There were pronounced differences from the fine structural aspects in late infantile cases. A large proportion of the inclusions did not exhibit a unit membrane. An hypothesis is proposed to clarify the delayed manifestation of this type of metachromatic leukodystrophy until childhood.

Topics: Acid Phosphatase; Adult; Brain; Demyelinating Diseases; Humans; Inclusion Bodies; Kidney Tubules; Kidney Tubules, Distal; Leukodystrophy, Metachromatic; Lysosomes; Macrophages; Male; Neurons; Peripheral Nerves; Schwann Cells; Sulfoglycosphingolipids

Topics: Acid Phosphatase; Animals; Arylsulfatases; Brain; Cerebrosides; Galactose; Glycolipids; Humans; Hydrogen-Ion Concentration; Leukodystrophy, Metachromatic; Male; Manganese; Mice; Sulfatases; Sulfoglycosphingolipids; Sulfuric Acids; Taurocholic Acid

In a family with a metachromatic leukodystrophy patient, two further pregnancies at risk were monitored by amnion cell culture. In one case, a normal baby was predicted and born. In the other case, a prenatal deficiency of arylsulfatase A was found. The diagnosis of metachromatic leukodystrophy was confirmed biochemically in various organs of the fetus by the deficiency of arylsulfatase A. The residual enzyme activity was shown to have an abnormal pH optimum and an increased heat stability. Ultrastructural studies revealed lipid storage in the myelinating nervous system and in the liver. For the interpretation of morphological results, it was indispensable to analyze an age-matched control fetus.

Topics: Arylsulfatases; Brain Chemistry; Cerebrosides; Female; Fetus; Humans; Hydrogen-Ion Concentration; Infant; Kidney; Leukodystrophy, Metachromatic; Lipid Metabolism; Liver; Nerve Fibers, Myelinated; Pregnancy; Prenatal Diagnosis; Spinal Cord; Sulfoglycosphingolipids; Temperature

Topics: Age Factors; Cerebroside-Sulfatase; Child; Female; Humans; Leukodystrophy, Metachromatic; Sulfoglycosphingolipids; Vitamin A

Topics: Age Factors; Child, Preschool; Female; Heterozygote; Homozygote; Humans; Leukodystrophy, Metachromatic; Phenols; Sulfatases; Sulfoglycosphingolipids

We compared the sphingolipid content of urine from a patient with Farber's disease with that of control urine. The ceramides were measured by high-performance liquid chromatography. The patient's urine contained 1.2 mug of ceramides per milligram of creatinine, more than 200-fold the normal amount. The urinary ceramides were isolated by high-performance liquid chromatography for further identification. They contained mainly nonhydroxy fatty acids and only a small quantity of those with 2-hydroxy fatty acids. This contrasts with the previously described composition of the patient's renal and cerebellar tissue. The fatty acid and long-chain base compositions of the urinary ceramides containing nonhydroxy fatty acids were nearly identical to those of the patient's kidney.

Topics: Ceramides; Cerebrosides; Cholesterol; Chromatography; Fatty Alcohols; Humans; Intellectual Disability; Kidney; Leukodystrophy, Metachromatic; Lipidoses; Lipids; Male; Pigmentation Disorders; Spectrophotometry, Infrared; Sphingomyelins; Sulfoglycosphingolipids

Myelin was isolated post mortem from brains of patients with metachromatic and globoid forms of leukodystrophy. In the leukodystrophies, isolates were obtained from central white matter and subcortical "U" fiber areas. Myelin also was obtained from age-matched human controls, and the method yielded light and heavy myelin fractions. In both disorders, the pathologic process affected the heavy isolates to a relatively greater degree. Comparative chemical analyses were made of the myelin isolates, and we concluded that a similar pathogenesis--the production of an unstable membrane in later stages of myelin formation--affects both disorders.

Topics: Cerebrosides; Cholesterol; Glycolipids; Humans; Infant; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Myelin Sheath; Nerve Tissue Proteins; Phospholipids; Sulfoglycosphingolipids

In a family with juvenile metachromatic leukodystrophy (sulfatide lipidosis) 2 patients showed residual arysulfatase A activities of 5--6%. The patients' healthy father was characterized biochemically by a 39% normal activity of leukocyte plus plasma arylsulfatase A. The father was further characterized by a high sulfatide excretion (0.2--0.5 mg/I urine) and, paradoxically, by a normal sulfatide degrading enzyme activity in vitro. This special carrier is suspected to be heterozygous for a) arylsulfatase A deficiency and b) arylsulfatase A (sulfatidase) lability. This presumed additional genetic defect could be the cause of the sulfatide excretion which, in turn, would be a sign of the preclinical stage of an exceptional form of adult metachromatic leukodystrophy. The normal sulfatidase activity seems to be due to an in vitro effect.

Topics: Adult; Arylsulfatases; Child; Drug Stability; Enzyme Activation; Female; Heterozygote; Humans; Leukodystrophy, Metachromatic; Male; Sulfatases; Sulfoglycosphingolipids

Topics: Age Factors; Autopsy; Cerebral Cortex; Cerebrosides; Chromatography, Gas; Chromatography, Thin Layer; Demyelinating Diseases; Fatty Acids; Histocytochemistry; Humans; Leukodystrophy, Metachromatic; Sulfoglycosphingolipids

Topics: Brain; Histocytochemistry; Humans; Kidney Tubules; Leukocytes; Leukodystrophy, Metachromatic; Methods; Staining and Labeling; Sulfoglycosphingolipids

Topics: Adult; Biopsy; Clinical Enzyme Tests; Humans; Leukocytes; Leukodystrophy, Metachromatic; Male; Sulfatases; Sulfoglycosphingolipids; Sural Nerve

Topics: Child; Chromatography, Thin Layer; Humans; Hydrolysis; Isoelectric Focusing; Kidney; Lactose; Leukodystrophy, Metachromatic; Staining and Labeling; Sulfatases; Sulfates; Sulfoglycosphingolipids

Topics: Coloring Agents; Electrophoresis, Polyacrylamide Gel; Humans; Hydrolases; Indicators and Reagents; Leukocytes; Leukodystrophy, Metachromatic; Sulfoglycosphingolipids

The livers of four patients with metachromatic leukodystrophy contained galactosyl sulfatide and lactosyl sulfatide, whereas these substances were undetectable in normal human liver. On the basis of methanolysis and permethylation studies, both sulfatides were shown to be substituted with sulfate at the C-3 position of the galactose moiety. Examination of the fatty acid compositions of these sulfatides showed that C(22:0) and higher 2-hydroxy and nonhydroxy fatty acids predominated in both. Both sulfatides contained the same long-chain bases, predominantly sphingosine, dihydrosphingosine, and phytosphingosine. Using as criteria the proportion of lactosyl sulfatide to galactosyl sulfatide, and the fatty acid and long-chain base compositions, the liver sulfatides from subjects with metachromatic leukodystrophy closely resemble those in the kidney and differ from those in brain and peripheral nerve.

Topics: Adolescent; Child, Preschool; Chromatography; Chromatography, Gas; Chromatography, Ion Exchange; Chromatography, Thin Layer; Drug Stability; Fatty Acids; Freezing; Galactose; Humans; Lactose; Leukodystrophy, Metachromatic; Liver; Male; Silicon Dioxide; Spectrophotometry, Infrared; Sulfoglycosphingolipids; Time Factors

Topics: Adult; Brain Chemistry; Cerebrosides; Cholesterol; Diffuse Cerebral Sclerosis of Schilder; Fatty Acids; Female; Frontal Lobe; Humans; Leukodystrophy, Metachromatic; Male; Middle Aged; Sphingolipids; Sphingomyelins; Sulfoglycosphingolipids

Topics: Child, Preschool; Gastric Mucosa; Humans; Leukodystrophy, Metachromatic; Male; Sulfoglycosphingolipids

Topics: Ceramides; Cerebrosides; Fabry Disease; Fucose; Galactosidases; Gaucher Disease; Glycolipids; Glycoside Hydrolases; Hexosaminidases; Humans; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Lipid Metabolism, Inborn Errors; Lipidoses; Neuraminic Acids; Niemann-Pick Diseases; Organ Specificity; Sphingomyelins; Sulfoglycosphingolipids

Topics: Cells, Cultured; Child; Child, Preschool; Culture Media; Fibroblasts; Histocytochemistry; Humans; Hydrogen-Ion Concentration; Inclusion Bodies; Leukodystrophy, Metachromatic; Lysosomes; Male; Microscopy, Electron; Sulfatases; Sulfoglycosphingolipids; Tolonium Chloride

Topics: Adolescent; Adult; Age Factors; Autopsy; Brain; Humans; Leukodystrophy, Metachromatic; Male; Microscopy, Electron; Occipital Lobe; Peripheral Nerves; Sulfoglycosphingolipids

Topics: Cerebrosides; Chromatography, Thin Layer; Coloring Agents; Humans; Leukodystrophy, Metachromatic; Methods; Sulfatases; Sulfoglycosphingolipids; Taurocholic Acid

The excretion of sulfatides in human urine was studied. 24-hr urine collections were filtered. Urinary glycolipids were extracted from the filter paper and fractionated on diethylaminoethyl cellulose and silicic acid columns, and by thin-layer chromatography. Fatty acids and long-chain bases were analyzed by gas-liquid chromatography of the corresponding esters and aldehydes. Glycosyl ceramide concentration was determined by gas-liquid chromatography of the trimethylsilyl ethers of the methyl glycosides. Normal females were found to excrete larger amounts of dihexosyl ceramides than males. Sulfatides were detected in all urine specimens. In sulfatidosis, a hereditary sulfatide storage disorder known as metachromatic leukcdystrophy, a large increase in sulfatide was readily apparent on a thin-layer chromatogram of the crude lipid extract. On comparing samples from normal individuals and patients with sulfatidosis, urinary sulfatide composition was remarkably similar to that previously reported in the kidney, including differences in fatty acid pattern. The determination of urinary sulfatides was a valuable confirmation of the deficiency in arylsulfatase A activity characteristic of sulfatidosis.

Topics: Adolescent; Adult; Aged; Cerebrosides; Child; Child, Preschool; Chromatography, Gas; Chromatography, Thin Layer; Fatty Acids; Female; Heterozygote; Hexoses; Humans; Infant; Kidney; Leukodystrophy, Metachromatic; Male; Middle Aged; Sulfatases; Sulfoglycosphingolipids

This paper describes the clinical, diagnostic, genetic and incidence aspects of globoid cell leukodystrophy and metachromatic leukodystrophy, the two least rare and most distinctive types among the leukodystrophies. It is based on experiences with 32 Swedish cases of globoid cell leukodystrophy (1953-1967) and 16 Swedish cases of metachromatic leukodystrophy (1956-1969). In both disorders it is now possible to make a correct diagnosis during life through various laboratory and biopsy procedures. An autosomal recessive inheritance is present in both of the disorders. The incidence of Krabbe's disease was found to be about 2:100,000 newborns in Sweden. The corresponding figure for metachromatic leukodystrophy is unknown at this time but is probably of about the same magnitude. This is the case in Northern Sweden.

Topics: Biopsy; Brain; Cerebrospinal Fluid Proteins; Child; Child, Preschool; Electroencephalography; Female; Humans; Infant; Kidney; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Liver; Male; Neural Conduction; Peripheral Nerves; Sulfoglycosphingolipids

Topics: Alkylation; Brain; Chromatography; Crystallization; Diffuse Cerebral Sclerosis of Schilder; Humans; Infant; Leukodystrophy, Metachromatic; Lipid Metabolism; Lipids; Sulfoglycosphingolipids; Tuberous Sclerosis

Topics: Humans; Leukodystrophy, Metachromatic; Lipidoses; Sulfoglycosphingolipids

Topics: Cerebrosides; Diffuse Cerebral Sclerosis of Schilder; Encephalitis; Humans; Leukodystrophy, Metachromatic; Lipidoses; Plasma; Sulfoglycosphingolipids