Deletion of fatty acid amide hydrolase reduces lyso-sulfatide levels but exacerbates metachromatic leukodystrophy in mice

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.     

Compound heterozygosity for metachromatic leukodystrophy and arylsulfatase A pseudodeficiency alleles is not associated with progressive neurological disease

Several allelic mutations at the arylsulfatase A (ASA) locus cause substantial deficiencies of this lysosomal enzyme. Depending on the genetically determined degree of the deficiency, the clinical outcome may be very different—either metachromatic leukodystrophy (MLD), a lethal lysosomal storage disorder affecting the nervous system, or, more frequently, the so-called pseudodeficiency (PD), which has no apparent clinical consequence. Because of compound heterozygosity for MLD and PD, 1/1,000 individuals in the population have low residual enzyme activities, which are intermediate between those of MLD patients and those of PD homozygous normal individuals. In order to assess whether PD/MLD compound heterozygotes bear a health risk, we examined clinically and biochemically 16 individuals with this genotype. Of these subjects, two had neurological symptoms and two showed lesions, without clinical symptoms, in magnetic resonance imaging of the brain. None of these symptoms was progressive, nor did they resemble those of MLD. Nerve conduction velocities were normal in these probands, and they secreted only low amounts of sulfatide in the urine. We conclude that the observed neurological symptoms are unrelated to the ASA genotype and that PD/MLD compound heterozygotes are not at an increased risk for developing progressive nervous system diseases.     

Lysosomal sulfoglycolipid storage in the kidneys of mice deficient for arylsulfatase A (ASA) and of double-knockout mice deficient for ASA and galactosylceramide synthase

The inherited deficiency of arylsulfatase A (ASA) causes lysosomal accumulation of sulfoglycolipids (mainly sulfo-galactosylceramide, S-GalCer ) and leads to metachromatic leukodystrophy in humans. Among visceral organs, kidneys are particularly affected. In the present study, the regional distribution and temporal development of sulfoglycolipid storage in kidneys of ASA-/- mice was investigated histochemically (alcian blue) and ultrastructurally. Furthermore, the sulfoglycolipid storage was examined in kidneys of double-knockout mice, which are incapable of: (a) degrading any sulfolipids (ASA-/-) and (b) synthesizing the major sulfolipid S-GalCer because of deficiency for galactosylceramide synthase (CGT), with the aim to search for additional ASA substrates. In ASA-/- mice, the nephron segments could be ranged in the order of decreasing sulfolipid storage: thin limbs of long-looped nephrons approximately thick ascending limbs > distal convoluted tubules > collecting ducts approximately short thin limbs. Macula densa and proximal tubules were unaffected. In ASA-/-/CGT-/- mice, the long thin limbs and distal convoluted tubules resembled those of ASA-/-/CGT+/+ mice, while the other segments showed less storage. The results suggest that the turnover of sulfolipids in general is highest in the distal nephron except macula densa, and that long thin limbs and distal convoluted tubules are the main sites for turnover of a minor sulfolipid species, which is known to be synthesized in the kidney of CGT-/- mice.     

Reduced brain cholesterol content in arylsulfatase A-deficient mice

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by deficiency in arylsulfatase A (ASA). Concentrations of cholesterol and its metabolites were determined in ASA deficient [ASA(-/-)] mice which serve as an animal model of MLD. We observed a significant reduction in cholesterol content in the brain of adult ASA(-/-) mice when compared to wild-type controls. This was not due to loss of myelin, because ASA(-/-) mice do not demyelinate. Other cholesterol metabolites were not changed significantly in ASA(-/-) mice, except for an increase in lathosterol. Moreover, reduced cholesterol levels were also found in tissue samples from two juvenile MLD cases. Since high cholesterol levels are important for myelination, and various cellular processes, like vesicular trafficking and signal transduction, reduced cholesterol levels might be an important factor in the molecular pathology of MLD.     

Genotype-phenotype relationship in various degrees of arylsulfatase A deficiency

Summary Arylsulfatase A (ASA) is a lysosomal enzyme that hydrolyzes sulfatide. Absence of ASA activity leads to metachromatic leukodystrophy (MLD). The clinical outcome resulting from ASA deficiency is highly variable with respect to age of onset and symptoms. So far the causes for the variability are poorly understood. We have studied the relationship between the ASA genotype and the clinical phenotype. Fibroblasts from a total of 34 subjects with low ASA activity were examined with immunoblotting, a sensitive ASA assay, and the sulfatide loading test in order to characterize low ASA activity further. By these methods, three different classes of ASA deficiency can be defined: homozygosity for the pseudodeficiency allele (ASA_P), compound heterozygosity for the ASA_P and MLD (ASA^–) alleles, and ASA^–/ ASA^– genotypes. These genotypes exhibit different levels of ASA residual activity. Only ASA^–/ASA^– genotypes are associated with MLD. For diagnostic purposes, however, the differentiation of the various ASA genotypes is essential.     

Sulfogalactosylceramides in motor and psycho-cognitive adult metachromatic leukodystrophy: relations between clinical, biochemical analysis and molecular aspects

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.     

Quantification of sulfatides and lysosulfatides in tissues and body fluids by liquid chromatography-tandem mass spectrometry

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.     

Molecular characteristics in Japanese patients with lipidosis: Novel mutations in metachromatic leukodystrophy and Gaucher disease

The characterization of mutations in Japanese patients with lipidosis, particularly in metachromatic leukodystrophy (MLD) and Gaucher disease has been studied in detail. Metachromatic leukodystrophy is characterized by an accumulation of sulfatide in nervous tissues and kidney due to a deficiency of arylsulfatase A (ASA). We analyzed the presence of three known mutant arylsulfatase A alleles in Japanese patients with MLD. Among 10 patients of Japanese patients with MLD, we found that allele 445A mutation has moderately high incidence and also homozygosity of this mutation results in the late infantile form. Allele 2381T was not found in Japanese patients. Furthermore, we found novel mutation which is G- to A mutation at the 1070 nucleotide of the ASA gene (designated 1070 A) in Japanese patients with juvenile onset. This mutation results in a amino acid substitution of Gly245 by Arg and found in heterozygote form. Our studies of molecular analysis in 10 Japanese patients with MLD indicate that Japanese MLD patients have unique characteristics of ASA mutations compared with those of Caucasian patients. On the other hand, Gaucher disease is the most prevalent sphingolipidosis, characterized by an accumulation of glucocerebroside in macrophage derived cells due to a deficiency of lysosomal hydrolase glucocerebrosidase. To study the molecular basis of Gaucher disease in Japanese patients, we analyzed the presence of the two known mutations (6433C and 3548A) in the glucocerebrosidase gene of 15 patients with Gaucher disease. We found that the 6433C and 3548A mutations occur in all subtypes of Japanese patients with Gaucher disease. Most frequent mutations among them was the 6433C mutation, 40% of 30 chromosomes, whereas the novel mutation of the 3548A found in Japanese patients with neuronopathic Gaucher disease was found in 20% (6 out of 30 chromosomes). The characteristics of these mutations in Japanese patients with Gaucher disease is different from those of Caucasian populations reported previously.     

An adult-type metachromatic leukodystrophy caused by substitution of serine for glycine-122 in arylsulfatase A

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease with autosomal recessive inheritance caused by a deficiency of the enzyme arylsulfatase A (ASA). We have identified a new mutation in the ASA gene of a patient with adult-type MLD. In this mutation, the glycine at position 122, a highly conserved residue in the AS gene family, was replaced by serine. In a transient expression study, COS cells transfected with the mutant cDNA carrying ¹²²GlySer did not show an increase of ASA activity and produced little material immunoreactive to an anti-ASA antibody, despite normal mRNA levels.     

A noninvasive mucinous cystic neoplasm with intermediate-grade dysplasia of the pancreas and extensive squamous metaplasia: a case report with clinicopathological correlation

?

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. Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1791578262610232

Patients and methods

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.

Results

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.

Conclusion

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.     

Pharmacokinetics and brain uptake in the rhesus monkey of a fusion protein of arylsulfatase a and a monoclonal antibody against the human insulin receptor

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder of the brain caused by mutations in the gene encoding the lysosomal sulfatase, arylsulfatase A (ASA). It is not possible to treat the brain in MLD with recombinant ASA, because the enzyme does not cross the blood‐brain barrier (BBB). In the present investigation, a BBB‐penetrating IgG‐ASA fusion protein is engineered and expressed, where the ASA monomer is fused to the carboxyl terminus of each heavy chain of an engineered monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via receptor‐mediated transport on the endogenous BBB insulin receptor, and acts as a molecular Trojan horse to ferry the ASA into brain from blood. The HIRMAb‐ASA is expressed in stably transfected Chinese hamster ovary cells grown in serum free medium, and purified by protein A affinity chromatography. The fusion protein retains high affinity binding to the HIR, EC50 = 0.34 ± 0.11 nM, and retains high ASA enzyme activity, 20 ± 1 units/mg. The HIRMAb‐ASA fusion protein is endocytosed and triaged to the lysosomal compartment in MLD fibroblasts. The fusion protein was radio‐labeled with the Bolton–Hunter reagent, and the [¹²⁵I]‐HIRMAb‐ASA rapidly penetrates the brain in the Rhesus monkey following intravenous administration. Film and emulsion autoradiography of primate brain shows global distribution of the fusion protein throughout the monkey brain. These studies describe a new biological entity that is designed to treat the brain of humans with MLD following non‐invasive, intravenous infusion of an IgG‐ASA fusion protein. Biotechnol. Bioeng. 2013; 110: 1456–1465. © 2012 Wiley Periodicals, Inc.     

In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice

Metachromatic leukodystrophy (MLD) is a lipidosis caused by deficiency of arylsulfatase A (ARSA). Although the genetics of MLD are known, its pathophysiology is not understood. The disease leads to progressive demyelination and early death and no effective treatment is available. We used lentiviral vectors to deliver a functional ARSA gene (human ARSA) into the brain of adult mice with germ-line inactivation of the mouse gene encoding ARSA, As2. We report sustained expression of active enzyme throughout a large portion of the brain, with long-term protection from development of neuropathology and hippocampal-related learning impairments. We show that selective degeneration of hippocampal neurons is a central step in disease pathogenesis, and provide evidence that in vivo transfer of ARSA by lentiviral vectors reverts the disease phenotype in all investigated areas. Therefore, in vivo gene therapy offers a unique option for MLD and other storage diseases affecting the central nervous system.     

Novel patient cell-based HTS assay for identification of small molecules for a lysosomal storage disease

Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as "plate fluorescence quencher" in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets.     

Exocytosis of storage material in a lysosomal disorder

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.     

Motor and psycho-cognitive clinical types in adult metachromatic leukodystrophy: genotype/phenotype relationships?

Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is biochemically characterized by an accumulation of sulfatides (sulfogalactosylceramides) mainly in oligodendrocytes and macrophages/microglia. The deficient enzyme is a lysosomal hydrolase, cerebroside sulfate sulfatase (arylsulfatase A). MLD is both a dysmyelinating and a demyelinating disease. The main clinical forms are infantile or juvenile, but some forms appear at adulthood. This disease involves also neuronal cells as sulfatides are also present in neurons in which the defect in degradation occurs also. We have studied 12 cases of adult MLD and clearly distinguished two clinical forms. One of them was characterized by mainly central nervous system motor signs (pyramidal, cerebellar, and seldom dystonia) and a peripheral neuropathy. The other form always started by behavioural abnormalities with modifications of mood, peculiar social reactions; a progressive mental deterioration occurred also. The diagnosis of schizophrenia was often mentioned. Most of these patients remained for many years without any neurological symptoms, and the diagnosis was only made when neurological signs appeared, or when Magnetic Resonance Imaging (MRI) was performed. MRI showed a diffuse demyelination, bilateral and often symmetrical, which could be temporarily limited to the periventricular areas. The diagnosis of adult MLD was biochemical, evidencing the low activity of arylsulfatase A (ASA) and sulfatide accumulation. To determine the respective participation of neurons and glial cells in the physiopathology of both the motor forms and the psycho-cognitive forms, our first approach was to search for mutations differing according to the clinical status. Motor forms involved the major adult ASA mutation P426L in a homozygote form in contrast to psycho-cognitive forms which involved as a compound heterozygote a specific I179S mutation.     

Brain Galactolipid Content in a Patient with Pseudo-Arylsulfatase A Deficiency and Coincidental Diffuse Disseminated Sclerosis, and in Patients with Metachromatic, Adreno-, and Other Leukodystrophies

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.     

An assay for the rapid detection of the arylsulfatase A pseudodeficiency allele facilitates diagnosis and genetic counseling for metachromatic leukodystrophy

Summary Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulfatase A (ASA). A substantial ASA deficiency has also been described in clinically healthy persons, a condition for which the term pseudodeficiency was introduced. The discrimination of both kinds of deficiencies based on ASA activity determination is difficult and unreliable. This creates a serious problem in the genetic counseling and diagnosis of MLD. The mutations characteristic for the pseudodeficiency (PD) allele have recently been identified. A non-radioactive assay based on the polymerase chain reaction is described, which allows the rapid detection of the ASA pd allele. The assay utilizes pairs of primers that allow either the amplification of the ASA PD allele or of other ASA alleles, since their 3 residues match either the ASA PD allele or other ASA alleles.     

Enhanced response to enzyme replacement therapy in Pompe disease after the induction of immune tolerance

Pompe disease, which results from mutations in the gene encoding the glycogen-degrading lysosomal enzyme acid alpha -glucosidase (GAA) (also called "acid maltase"), causes death in early childhood related to glycogen accumulation in striated muscle and an accompanying infantile-onset cardiomyopathy. The efficacy of enzyme replacement therapy (ERT) with recombinant human GAA was demonstrated during clinical trials that prolonged subjects' overall survival, prolonged ventilator-free survival, and also improved cardiomyopathy, which led to broad-label approval by the U.S. Food and Drug Administration. Patients who lack any residual GAA expression and are deemed negative for cross-reacting immunologic material (CRIM) have a poor response to ERT. We previously showed that gene therapy with an adeno-associated virus (AAV) vector containing a liver-specific promoter elevated the GAA activity in plasma and prevented anti-GAA antibody formation in immunocompetent GAA-knockout mice for 18 wk, predicting that liver-specific expression of human GAA with the AAV vector would induce immune tolerance and enhance the efficacy of ERT. In this study, a very low number of AAV vector particles was administered before initiation of ERT, to prevent the antibody response in GAA-knockout mice. A robust antibody response was provoked in naive GAA-knockout mice by 6 wk after a challenge with human GAA and Freund's adjuvant; in contrast, administration of the AAV vector before the GAA challenge prevented the antibody response. Most compellingly, the antibody response was prevented by AAV vector administration during the 12 wk of ERT, and the efficacy of ERT was thereby enhanced. Thus, AAV vector-mediated gene therapy induced a tolerance to introduced GAA, and this strategy could enhance the efficacy of ERT in CRIM-negative patients with Pompe disease and in patients with other lysosomal storage diseases.     

Accumulation of sulfatide in neuronal and glial cells of arylsulfatase A deficient mice

Arylsulfatase A (ASA) degrades sulfatide, seminolipid and lactosylceramide sulfate, glycolipids recognized by the Sulph I antibody although sulfatide is considered the main antigen. Sulfatide is myelin associated but studies have shown a minor distribution also in non-myelin forming cells. The aim of this work was to further study sulfatide in neurons and astrocytes by immunohistochemistry, facilitated by investigation of tissue from adult ASA deficient (ASA ?/?) mice. Cells with a low presence of sulfatide might be detected due to lack of ASA activity and accumulation of Sulph I antigens. Sulfatide positive astrocytes and neurons were more numerous and intensely stained in ASA ?/? mice, demonstrating a sulfatide accumulation compared to controls. Sulph I staining was especially increased in the molecular layer of cerebellum, in which Purkinje cell dendrites displayed an altered morphology, and in layer IV–VI of cerebral cortex. In hippocampus, immunostaining was found in neuronal cytoplasm in ASA ?/? but in nuclear membranes of control mice. We observed a gray matter astrogliosis, which appeared to be associated to sulfatide accumulation. In addition, the developmental change (<20 months) of Sulph I antigens, galactosylceramide, phospholipids and cholesterol were followed by lipid analyses which verified sulfatide and seminolipid accumulation in adult ASA ?/? mice, although no lactosylceramide sulfate could be detected. In addition to demonstrating sulfatide in neurons and astrocytes, this study supports the value of ASA ?/? mice as a model for metachromatic leukodystrophy and suggests that accumulation of sulfatide beyond myelin might contribute to the pathology of this disease.     

Characterization of a recombinant molecule covalently indistinguishable from human cerebroside-sulfate activator protein (CSAct or Saposin B).

Humans deficient in the cerebroside-sulfate activator protein (CSAct or Saposin B) are unable to catabolize sulfatide and other glycosphingolipids leading to their accumulation and neurodegenerative disease. Clinically this usually manifests as a form of metachromatic leukodystrophy (MLD). CSAct is a small water-soluble glycoprotein that apparently functions in the lysosome to solubilize sulfatide and other lipids enabling their interaction with soluble lysosomal hydrolases. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A or ex vivo by its ability to functionally complement CSAct deficient fibroblast cell lines derived from MLD patients. A recombinant form of CSAct has been expressed in E. coli and processed in vitro to a form covalently indistinguishable from deglycosylated human CSAct isolated from human urine. Size-exclusion chromatography in combination with multi-angle laser-light scattering (SEC-MALLS) measurements demonstrate that both native and recombinant forms of the molecule behave as a dimer in the pH range 7.0-4.5. The CSAct activity assay showed that both recombinant and deglycosylated human urine CSAct efficiently activated sulfatide sulfate hydrolysis and provided functional complementation of CSAct-deficient cells. However, a D21N mutant form of recombinant CSAct could not functionally complement these cells despite full activity in the in vitro assay. It is concluded that while glycosylation is unnecessary for in vitro and ex vivo activity of CSAct, modification of the native N21 is necessary to prevent loss of ex vivo activity, possibly via protection from degradation.