Mucolipidosis type III (ML III, pseudo-Hurler polydystrophy), an autosomal recessive inherited disorder of lysosomal enzyme targeting is due to a defective N-acetylglucosamine 1-phosphotransferase (phosphotransferase) activity and leads to the impaired formation of mannose 6-phosphate markers in soluble lysosomal enzymes followed by their increased excretion into the serum. Mutations in the phosphotransferase gamma subunit gene (GNPTAG) have been reported to be responsible for ML III. Here we report on a 14-year-old adolescent with a mild clinical phenotype of ML III. He presented with progressive joint stiffness and swelling. Urinary oligosaccharide and mucopolysaccharide excretion was normal. Lysosomal enzyme activities were significantly elevated in the serum and decreased in cultured fibroblasts. Impaired trafficking of the lysosomal protease cathepsin D (CtsD) was confirmed by metabolic labeling of the patient's fibroblasts. Neither mutations in the GNPTAG gene nor alterations in the GNPTAG mRNA level were detected whereas the steady state concentration of the 97 kDa GNPTAG dimer was reduced. Most importantly, the patient is homozygous for a pathogenic nucleotide substitution and a polymorphism in the phosphotransferase alpha/beta subunit gene (GNPTA). The data indicate that defects in genes other than GNPTAG can be linked to ML III contributing to the variability of the phenotype.

We report the case of a 27-year-old female with recurrent paranoid-hallucinatory episodes who was initially diagnosed as suffering from schizophrenic psychosis. After 10 years of treatment under this diagnosis, alpha-mannosidosis was identified to be the underlying cause of her psychiatric symptoms. alpha-Mannosidosis is a rare autosomal recessive lysosomal storage disorder associated with decreased activity of the enzyme mannosidase. In the present case, diagnosis was made late in the illness after failure of a response to antipsychotic treatment and with the patient additionally showing progressive cognitive decline. Only after extensive investigation was the diagnosis made by showing decreased alpha-mannosidase enzyme activity in serum and blood leukocytes. This case demonstrates that an unusual clinical course or striking symptom patterns, especially in association with somatic comorbidity, in psychotic patients should lead to diagnostic consideration of inherited metabolic disease.

Wir berichten über eine 27-jährige Patientin, bei der nach 10-jährigem Krankheitsverlauf mit rezidivierenden paranoid-halluzinatorischen Episoden und Behandlung unter der Diagnose einer schizophrenen Psychose eine α-Mannosidose als Ursache der psychiatrischen Symptome gesichert werden konnte. Die α-Mannosidose ist eine seltene autosomal-rezessiv vererbte lysosomale Speichererkrankung mit verminderter Aktivität des Enzyms α-Mannosidase. Im vorliegenden Fall wurde die Diagnose erst relativ spät nach therapierefraktärem Behandlungsverlauf und zusätzlichem Auftreten einer progredienten kognitiven Störung gestellt. Erst nach umfangreicher differenzialdiagnostischer Abklärung wurde über den Nachweis einer verminderten α-Mannosidase-Aktivität in Serum und Leukozyten die Diagnose etabliert. Diese Kasuistik veranschaulicht, dass außergewöhnliche klinische Verläufe oder auffällige Symptomkonstellationen, insbesondere in Verbindung mit organischer Komorbidität, bei psychotischen Patienten zu differenzialdiagnostischen Überlegungen in Richtung hereditärer Stoffwechselerkrankungen Anlass geben sollten.

alpha-Mannosidosis is an inherited lysosomal storage disease. The authors report three siblings ( ages 38 to 47 years) with the rare adult variant. All three had late-onset ataxia and retinal degeneration, adding to hearing loss, cognitive impairment, and dysotosis multiplex. One sibling also had psychosis. MRI revealed cerebellar atrophy and predominantly parieto-occipital white matter changes. MR spectroscopy showed no evidence for demyelination. It appears that the disabling course of adult alpha-mannosidosis is caused by lysosomal accumulation rather than demyelination.

The definitive diagnosis of lysosomal storage disorders depends on the determination of enzymatic activities in cells, tissues or body fluids. At present, neither an evaluation of the different methods nor an interlaboratory quality assurance scheme is available. We have therefore determined the activities of total hexosaminidase, hexosaminidase A and β-galactosidase in the same samples (n=15) at two metabolic centres in Germany. Three different enzymatic methods were employed, two of which were based on leukocytes as enzyme source and one on dried blood spots. The results obtained by the two different methods using leukocytes proved comparable. In contrast, assays with dried blood spots showed poor correlation with results from leukocytes, possibly because enzymatic activity in dried blood is mainly derived from soluble plasma proteins. Nevertheless, accurate detection of a true enzyme deficiency was also possible in dried blood spots. All enzymes were highly stable when mailed frozen (recovery 98–120%). Enzymatic activities in dried blood samples were also stable at room temperature and were not affected even by exposure to elevated temperatures (50°C for 3h). Dried blood seems to be especially well suited for mailing from distant healthcare facilities, although more accurate results can be expected from leukocytes. In summary, comparability and pitfalls within a lysosomal quality assurance programme were evaluated.

The SK-N-MC cell line is frequently used as a model of neuronal differentiation induced by 5-bromodeoxyuridine (BrdU). In this study, the differentiation properties of this cell line were investigated under hydroxyl free radical generation, and compared to BrdU treatment. Hydroxyl free radicals were generated in the cultures by the Fenton reaction, i.e. by simultaneous addition of ADP-Fe2+ complex and H2O2. Microscopic morphological signs, as well as the acetylcholinesterase and ganglioside sialidase activities were considered as markers of neuronal differentiation of this cholinergic neuroblastoma cell line. Apart from the altered morphological appearance, the marker enzymes displayed significant increases after both types of intervention. We suggest that hydroxyl free radicals can induce in vitro cell differentiation. They apparently play a more complex role in cell physiology than simply causing oxidative damage.

The deficiency of the lysosomal neuraminidase (NEU1; sialidase) causes the storage disorder sialidosis with symptoms ranging from eye abnormalities and neurological disturbances to skeletal malformations, mental retardation and early death. Sialidosis patients encompassing a wide spectrum of clinical symptoms were screened for mutations in neu1. We identified the same homozygous interstitial deletion (11 kb) in two patients causing the fusion of exon 10 of CTL4 (New Gene 22; NG22) with the 3'-UTR of neu1. In one patient we found the resulting CTL4/Neu1 fusion transcript, in the other we detected an alternatively spliced CTL4 transcript (retention of intron 9).

Gangliosides located in the outer leaflet of the plasma membrane are important modulators of cellular functions. Our previous work has shown that in cultured human SK-N-MC neuroblastoma cells a sialidase residing in the same membrane selectively desialylates gangliosides with terminal sialic acid residues, causing a shift from higher species to GM1 and a conversion of GM3 to lactosylceramide. Inhibition of this sialidase by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en) resulted in increased cell proliferation and a loss of differentiation markers. In this study, we examined the occurrence and function of this ganglioside sialidase in other neuronal cells. Subcellular fractionation showed the sialidase to be located in the plasma membrane of all cell lines studied. The presence of the inhibitor NeuAc2en led to a profound decrease in the amount of the differentiation marker 200 kDa/70 kDa neurofilaments and an increase in cell proliferation in the cholinergic SK-N-MC and mixed cholinergic/adrenergic SK-N-FI and SK-N-DZ neuroblastoma lines, but had little or no effect in the human adrenergic SK-N-SH and SK-N-AS and the adrenergic/cholinergic PC12 cells from rat. The influence of the inhibitor on cell behaviour was paralleled by a diminished number of cholera toxin B-binding GM1 sites. The findings demonstrate that the plasma membrane ganglioside sialidase is an important element of proliferation and differentiation control in some, but not all, neuroblastoma cells and suggest that there might be a relationship between plasma membrane sialidase activity and cholinergic differentiation.

Gangliosides of the plasma membrane are important modulators of cellular functions. Recent reports have shown their enrichment in glycosphingolipid-containing membrane microdomains, called glycosphingolipid-signaling domain or rafts, which can be isolated due to their insolubility in Triton X-100 and flotation through a sucrose gradient. In previous work on neuroblastoma cells we had found that a ganglioside-specific sialidase activity of the plasma membrane controlled proliferation and differentiation through selective ganglioside desialylation. Assuming the ganglioside sialidase to be close to its substrates in the membrane, we investigated its association with detergent-insoluble microdomains in the neuroblastoma cell line SK-N-MC. The results show that the ganglioside sialidase codistributes with the raft markers ganglioside GM1, flotillin, src family kinases, and glycosylphosphatidylinositol-anchored proteins in a fraction containing about 2% of cellular protein. The association of the ganglioside sialidase with glycosphingolipid-enriched membrane fractions therefore is in support of a role of this glycosidase in ganglioside-dependent signaling processes.

The orientation of the catalytic site of a ganglioside-specific sialidase in the plasma membrane of SK-N-MC neuroblastoma cells was probed using water-soluble GD1a-neoganglioprotein substrate on intact cells and GM1-product detection by cholera toxin B. Desialylation of substrate was readily observed, whereas specific sialidase inhibitors prevented the reaction, and conditioned medium was inactive. Inhibitors of endocytosis and acidification had no effect on substrate degradation, and lowering temperature to 18 degrees C reduced activity but did not abolish it. We conclude that the ganglioside sialidase activity is cell surface-orientated and displays an in situ specificity that mirrors enzyme preparations in vitro.

The cell density-dependent growth inhibition of human SK-N-MC neuroblastoma cells is initiated by increased ganglioside sialidase activity leading to elevated cell surface presentation of ganglioside GM1, a ligand of galectin-1. We herein show that the extent of the cell surface expression of the galectin coincides with marked increases of the sialidase activity. Reverse transcriptase-polymerase chain reaction analysis excludes a regulation at the transcriptional level. Exposure of cells to purified galectin-1 reveals its carbohydrate-dependent activity to reduce cell proliferation. Assays to detect DNA fragmentation biochemically and cytometrically and to block caspases render it unlikely that galectin-1 acts as a classical proapoptotic factor on these cells. Because the chimeric galectin-3 shares binding sites and binding parameters with galectin-1 for these cells, we tested whether this galectin will elicit the same response as the homodimeric cross-linking galectin-1. Evidently, galectin-3 fails to affect cell growth by itself but interferes with galectin-1 upon coincubation. Its proteolytically truncated variant, the C-terminal lectin domain with impaired capacity to form aggregates when surface bound, has only weak binding properties. Thus, the way in which the galectin-1 interacts topologically with an apparently common set of ligands relative to galectin-3 is crucial for eliciting post-binding events. We conclude that galectin-1 is a probable effector in the sialidase-dependent growth control in this system. Moreover, the experiments with galectin-3 reveal functional divergence, most probably based on different topologies of presentation of homologous carbohydrate-binding sites.

Sialidosis is a lysosomal storage disease caused by the deficiency of alpha-N-acetylneuraminidase (NEU1; sialidase), the key enzyme for the intralysosomal catabolism of sialylated glycoconjugates. We have identified a homozygous transversion in the last intron (IVSE +1 G>C) in neu1 of a sialidosis patient. Sequencing of the truncated cDNA revealed an alternatively spliced neu1 transcript which lacks the complete sequence of exon 5. Skipping of exon 5 leads to a frameshift and results in a premature termination codon. This is the first description of an intronic point mutation causing a complete deficiency of the lysosomal neuraminidase activity.

Syrian newborn with coarse facies, hepato-splenomegaly, and refractory ascites is reported. Examination of the ascitic fluid showed vacuolated lymphocytes and thin-layer chromatography of urinary oligosaccharides revealed an abnormal pattern indicative of sialidosis. Despite intensive care, the baby died of respiratory insufficiency 28 days after birth. In cultured skin fibroblasts an increase of the incorporation of [C-14]methylamine pointed to excessive lysosomal storage and the demonstration of an isolated deficiency of alpha-N-acetylneuraminidase (sialidase) led to the diagnosis of a sialidosis. At postmortem examination, foam cells were found mostly in bone marrow, liver, and brain. To date very few cases of neonatal sialidosis have been reported, and, to the best of our knowledge, this is the first child with neonatal sialidosis from Syria and the first case of neonatal sialidosis studied by the [C-14]methylamine incorporation assay.

Cell density-dependent inhibition of growth and neural differentiation in the human neuroblastoma cell line SK-N-MC are associated with a ganglioside sialidase-mediated increase of GM(1) and lactosylceramide at the cell surface. Because these glycolipids expose galactose residues, we have initiated the study of the potential role of galectins in such cellular events. Using specific antibodies, galectin-1 but not galectin-3 was found to be present at the cell surface. Assessment of carbohydrate-dependent binding revealed a saturable amount of ligand sites approaching 2.6 x 10(6) galectin-1 molecules bound/cell. Presence during cell culture of the sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid or of the GM(1)-binding cholera toxin B subunit effected a decrease of the presentation of galectin-1 ligands by 30-50%. The assumption that GM(1) is a major ligand for galectin-1 was reinforced by the correlation between the number of carbohydrate dependent I-125-iodinated GM(1)-neoganglioprotein binding sites and the amount of immunoreactive surface galectin-1, the marked sensitivity of probe binding to the presence of anti-galectin-1 antibody, and the inhibition of cell adhesion to surface-immobilized GM(1) by the antibody. The results open the possibility that the carbohydrate-dependent interaction between ganglioside GM(1) and galectin-(1) may relay sialidase-dependent alterations in this cell system.

Niemann-Pick disease type C is a panethnic autosomal recessive neurovisceral storage disorder characterised by a unique error in cellular trafficking of exogenous cholesterol. The most typical phenotype is characterised by hepatosplenomegaly, vertical supranuclear ophthalmoplegia, progressive ataxia, dystonia, and dementia and manifests in late childhood. Adult onset Niemann-Pick disease type C often includes psychosis and dementia.1 2 We report an adult female patient with biochemically verified Niemann-Pick disease type C in whom the initial diagnosis of multiple sclerosis was questionable because of a persistent splenomegaly. The 33 year old woman was referred to the Psychiatry Department of the University of Heidelberg in December 1994. She complained of progressive difficulties with memory and concentration, increasing social isolation, and problems with daily activities. The patient was unsure about the onset of her problems but suggested that they had started after the birth of her daughter three years previously. History, family history, and the patient’s fetal and childhood development were unremarkable. Her parents are non-consanguineous. School performance was satisfactory initially but later her learning behaviour became erratic and she failed her examinations after 10 school years. She worked as a saleswoman and after giving birth she lived on social support. With time she no longer managed to educate …

Gangliosides, constituents of surfaces of vertebrate cells, modulate important cellular functions. Ganglioside-specific sialidases that possibly control these processes have been observed in a number of tissues, but their characterization has proved difficult due to their low abundance and lability. Here we describe the partial isolation and characterization of a ganglioside sialidase from human brain grey matter. After membrane extraction with octylglucoside, the enzyme was purified about 1300-fold by ion-exchange, affinity and gel-permeation chromatographies. Although PAGE still showed several protein bands, specific photoaffinity labelling with iodinated 5-N-acetyl-9-(4-azidosalicoylamido)-2,9-dideoxy-2,3-didehydrone uraminic acid identified a single polypeptide of 60 kDa likely to contain the active site of the sialidase. In the presence of 0.4% octylglucoside, the purified sialidase desialylated gangliosides G(M3), G(D1a), G(D1b) and G(T1b), but was inactive towards G(M1), G(M2), colominic acid, sialyl-(alpha2-3)-lactose, 2-(4-methylumbelliferyl)-neuraminate, or the glycoprotein fetuin. The ganglioside sialidase activity was strongly inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, heparin and heparan sulfate. Because of its substrate and inhibitor profiles, the purified enzyme resembles the activity characterized previously in the plasma membrane of human neuroblastoma cells, but is distinct from a lysosomal activity. The purified brain sialidase thus appears to function in the selective desialylation of gangliosides with terminal sialic acid residues.

Gangliosides on the external side of the plasma membrane are important modulators of cellular functions. In previous work we had found that in cultured human SK-N-MC neuroblastoma cells a cell surface sialidase activity specifically cleaved terminal sialic acids from gangliosides, leading to a shift from higher sialylated species to GM1 and a decrease of GM3. To further elucidate the function of the enzyme, we have now examined the consequences of ganglioside sialidase inhibition. When present in the culture medium, the ganglioside sialidase inhibitors 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en), heparin, and heparan sulfate caused dramatic changes in cell behavior. Thus, the inhibitors uniformly led to a complete release from contact inhibition of growth, and to the loss of the differentiation markers neuron-specific enolase and neurofilaments, and a decrease of cyclic AMP. In presence of NeuAc2en, cells that normally were spread out evenly and were firmly attached, appeared smaller, rounded, and only loosely adherent to the culture vessel. Exogenous addition of vibrio cholerae sialidase mimicked the action of the plasma membrane ganglioside sialidase by retarding cell proliferation and increasing intracellular acetylcholinesterase. That the ganglioside sialidase inhibitors in the culture medium indeed affected solely the cell surface enzyme and not also a lysosomal sialidase, was demonstrated in an experiment where the desialylation of exogenously added radioactive gangliosides was determined in absence and presence of NeuAc2en and NH4Cl, an inhibitor of lysosomal function. Taken together, our results suggest that the ganglioside sialidase on the surface of SK-N-MC cells is responsible for growth control and differentiation in this neuronal cell line.

Tay-Sachs disease is a genetically determined neurodegenerative disorder, resulting from mutations of the hexosaminidase (Hex) A gene coding for the alpha-subunit of beta-D-N-acetyl-hexosaminidase. Clinically, there is severe encephalomyelopathy leading to death within the first few years of life. Hex A activity is usually absent in tissue and body fluids of these patients. Juvenile and adult Hex A deficiencies are less severe but rare variants with some residual Hex A activity. All these variants are most prevalent among Ashkenazi Jews. We describe a non-Jewish family in which four adult brothers and sisters had markedly reduced Hex A activities and onset of symptoms in the second decade of life. The phenotypical expression was remarkably homogeneous, consisting in a combination of slowly progressive motor neuron disease, ataxia and ocular motor disturbances. None of the patients were demented at this stage of their illness. Magnetic resonance studies showed severe cerebellar atrophy, but were otherwise normal. Hex A deficiency was established by biochemical measurements in the serum and skin fibroblasts using the fluorogenic substrates 4-MUG and 4-MUGS as well as by gel electrophoresis. Molecular genetic studies revealed that the patients are compound heterozygotes for the 'adult' mutation Gly269 --> Ser and the 'infantile' 4-base insertion in exon 11 of the Hex A gene.