Variations of the ultrastructure of neuronal lipofuscin during childhood and adolescence in the human Ammon's horn.

The ultrastructure of lipofuscin (Lf) was studied in hippocampal and neocortical neurons of children and youngsters between 3 months and 24 years of age. As a standard, regions CA1 and CA4 of Ammon's horn and the gyrus centralis anterior of the left hemisphere were examined, and the ratio of the two components of Lf, the pigment part, and the usually droplet-like lipid part was looked at. Few and small granules with typical linear structures in the pigment part and little lipid droplets were found as early as at the age of 3 months in all brain regions. There were no morphological differences of Lf in the areas of Ammon's horn up to 3 years, but the Lf ultrastructure in Ammon's horn differed clearly from that in the neocortical region. Differences of Lf between the areas CA1 and CA4 were found to appear at the age of 6-8 years, to have a rather variable pattern between age 11 and about 20 years, and to be relatively constant thereafter. The Lf pigment part consisted of irregularly arranged three laminar linear structures. Some varieties could be seen in the size and shape of the Lf granules and in the lipid/pigment ratio. As to the question of Lf being an "age pigment," the findings that the number of Lf granules did not further increase after the period of early adolescence was not consistent with the age pigment hypothesis. No regional or age-dependent differences were found in the Lf of astro- and oligodendroglia.

The role of ceramide in receptor- and stress-induced apoptosis studied in acidic ceramidase-deficient Farber disease cells.

The activation of sphingomyelinases leading to the generation of ceramide has been implicated in various apoptotic pathways. However, the role of ceramide as an essential death mediator remains highly controversial. In the present study, we investigated the functional relevance of ceramide in a genetic model by using primary cells from a Farber disease patient. These cells accumulate ceramide as the result of an inherited deficiency of acidic ceramidase. We demonstrate that Farber disease lymphocytes and fibroblasts underwent apoptosis induced by various stress stimuli, including staurosporine, anticancer drugs and gamma-irradiation, equally as normal control cells. In addition, caspase activation by these proapoptotic agents occurred rather similarly in Farber disease and control fibroblasts. Interestingly, Farber disease lymphoid cells underwent apoptosis induced by the CD95 death receptor more rapidly than control cells. Our data therefore suggest that ceramide does not play an essential role as a second messenger in stress-induced apoptosis. However, in accordance with a role in lipid-rich microdomains, ceramide by altering membrane composition may function as an amplifier in CD95-mediated apoptosis.

Immunocytochemical localization of sphingolipid activator protein 2 (SAP-2) in normal and SAP-deficient fibroblasts.

The intracellular localization of sphingolipid activator protein 2 (SAP-2) was determined immunocytochemically using an antiserum raised against a SAP-2 preparation from Gaucher spleen. The immunolabeling indicated that SAP-2 was largely localized in the lysosomes of fibroblasts from normal individuals. In some lysosomes the labeling was greatest around the perimeter of the matrix, suggesting an association between the activator and lysosomal membrane components. The labeling technique was also applied to fibroblasts from a patient with a unique sphingolipid storage disorder. Consistent with immunoblotting studies on electrophoretograms, both the patient and his affected fetal sibling were found to be deficient in immunoreactive SAP-2.

Saposins (sap) A and C activate the degradation of galactosylsphingosine.

As previously shown for [(3)H-galactosyl]ceramide, the breakdown of [(3)H-galactosyl]sphingosine was reduced in prosaposin-deficient skin fibroblast homogenates. Galactosylsphingosine hydrolysis was also deficient in cell homogenates from Krabbe's disease (beta-galactocerebrosidase-deficient) patients, but not acid beta-galactosidase-deficient patients. Moreover, hydrolysis of galactosylsphingosine in the prosaposin-deficient cell homogenates could be partially restored by adding pure saposin A or C, thereby identifying these saposins as essential facilitators of galactosylsphingosine hydrolysis. By contrast, saposins B and D had little effect on galactosylsphingosine hydrolysis in the prosaposin-deficient cells. The reduced galactosylsphingosine turnover in prosaposin-deficiency suggests that there could be a pathogenetic cerebral accumulation of galactosylsphingosine in this disorder.

Saposins (sap) A and C activate the degradation of galactosylceramide in living cells.

In loading tests using galactosylceramide which had been labelled with tritium in the ceramide moiety, living skin fibroblast lines derived from the original prosaposin-deficient patients had a markedly reduced capacity to degrade galactosylceramide. The hydrolysis of galactosylceramide could be partially restored in these cells, up to about half the normal rate, by adding pure saposin A, pure saposin C, or a mixture of these saposins to the culture medium. By contrast, saposins B and D had little effect on galactosylceramide hydrolysis in the prosaposin-deficient cells. Cells from beta-galactocerebrosidase-deficient (Krabbe) patients had a relatively high residual galactosylceramide degradation, which was similar to the rate observed for prosaposin-deficient cells in the presence of saposin A or C. An SV40-transformed fibroblast line from the original saposin C-deficient patient, where saposin A is not affected, showed normal degradation of galactosylceramide. The findings support the hypothesis, which was deduced originally from in vitro experiments, that saposins A and C are the in vivo activators of galactosylceramide degradation. Although the results with saposin C-deficient fibroblasts suggest that the presence of only saposin A allows galactosylceramide breakdown to proceed at a normal rate in fibroblasts, it remains to be determined whether saposins A and C can substitute for each other with respect to their effects on galactosylceramide metabolism in the whole organism.

Natural ceramide is unable to escape the lysosome, in contrast to a fluorescent analogue.

Since the generation upon cell stimulation of the second messenger ceramide has been reported to occur in an endosomal/lysosomal compartment, we investigated whether ceramide formed in the lysosomes can escape this compartment. The metabolic fate of radiolabelled ceramide produced by intralysosomal hydrolysis of LDL-associated [ceramide-3H]sphingomyelin or [stearoyl-1-(14)C]sulfatide was examined in fibroblasts from control individuals and a patient with inborn lysosomal ceramidase deficiency (Farber disease). The behavior of this radioactive ceramide was compared to that of a fluorescent (lissamine-rhodaminyl) ceramide analogue deriving from sulfatide degradation. While in Farber cells the natural, radiolabelled ceramide remained completely undegraded and accumulated in the lysosomes, the fluorescent derivative was rapidly converted to sphingomyelin. These findings strongly suggest that, in contrast to fluorescent derivatives, endogenous long-chain ceramide is unable to exit from lysosomes, therefore making the lysosomal ceramide unlikely to be a biomodulatory molecule.

Significance of lipopigments with fingerprint profiles in eccrine sweat gland epithelial cells.

Lipopigments with fingerprint profiles in eccrine sweat gland epithelial cells are regular findings in childhood NCL. They have also been described in adult NCL (ANCL) a few times, but not consistently. However, they have been considered nonspecific when not matched by similar abnormal profiles in noneccrine sweat gland epithelial cells. These conflicting reports may pose a diagnostic dilemma as outlined in the following 2 examples. Patient 1 is a 20-year-old man who developed severe tetraparesis and dementia over 2 years. Electroencephalogram was abnormal with epileptiform discharges. The patient died at age 21 years without autopsy; no other relatives are known to have a similar disease. Patient 2, a 49-year-old woman, developed ataxia and gait abnormalities when 44 years old, and, later, psychosis and dementia. The patient is still alive; no other family members are similarly affected. Both patient lacked evidence of a retinopathy clinically, funduscopically, and by electroretinography. Both patients showed lipopigments within secretory eccrine sweat gland epithelial cells which harbored unequivocal fingerprint profiles, but not within noneccrine sweat gland cells. Regular lipofuscin was observed in other cells, including skeletal muscle fibers of patient 2.

Methylamine accumulation in cultured cells as a measure of the aqueous storage compartment in the laboratory diagnosis of genetic lysosomal diseases.

Intracellular accumulation of the lysosomotropic compound [14C]methylamine was used to estimate the size of the lysosomal compartment in fibroblasts cultured from patients with a variety of lysosomal storage diseases. In previous work from our laboratory, it was shown that methylamine accumulation was significantly increased in diseases with infantile or juvenile onset and storage of predominantly water-soluble material such as in the mucopolysaccharidoses, mucolipidoses, and oligosaccharidoses. In the present study, methylamine incorporation was abnormally increased in cells from patients with glycogenosis type II and with Niemann-Pick type C disease, whereas it was normal in other sphingolipidoses and in the late-infantile and juvenile forms of neuronal ceroid lipofuscinoses. The methylamine test was also checked regarding its potential use for prenatal diagnostic testing. In model systems with cultured amniotic or chorionic villus cells, lysosomal storage was experimentally induced by the cathepsin inhibitor leupeptin and was readily detected when compared to untreated controls. Cultured amniotic cells from a fetus with mucopolysaccharidosis II were found to incorporate significantly higher amounts of [14C]methylamine than the normal controls. The results indicate that the methylamine accumulation method is an additional tool in the diagnosis and prenatal diagnosis of lysosomal diseases with abnormal storage of water-soluble material.

Leukodystrophy incidence in Germany.

Through a survey of all departments of pediatrics, neurology and neuropathology in Germany, we calculated the incidence of all major forms of leukodystrophy. Only diagnoses based on specific biochemical tests in association with typical findings and/or neuroradiologically proven white matter involvement were accepted. In accordance with these strict criteria, 617 cases of leukodystrophy were found (incidence of all forms: app. 2.0/100,000). Minimal incidence was estimated at 0.8/100,000 for adrenoleukodystrophy/adrenomyeloneuropathy (ALD/AMN), 0.6/100,000 for metachromatic leukodystrophy (MLD), and 0.6/100,000 for Krabbe disease. Thus ALD/AMN is apparently underdiagnosed in Germany. A considerable proportion of leukodystrophies could not be classified in spite of adequate diagnostic procedures in experienced centers.

Adrenoleukodystrophy in an adult female. A clinical, morphological, and neurochemical study.

A 43-year-old female with adrenoleukodystrophy (ALD) is described, who developed spastic tetraparesis, suffered grand mal seizures, and became stuporous and demented during the last 5 years of her life. Computed tomography revealed symmetrical hypodense lesions in the peritrigonal regions. Adrenal insufficiency was not evident except for skin pigmentation. The ultrastructure of a rectal biopsy specimen showed inclusions with lamellae and interspersed clefts in macrophages of the submucosal layer. At autopsy, the adrenals were found to contain large foam cells filled with similar inclusions. The brain cortex and the spinal cord were histologically normal. However, cerebral white matter exhibited widespread demyelination which spared only the arcuate fibres. In regions of less severe demyelination scattered inflammatory cells were seen. On electron microscopy, aggregates of typical paired leaflets with distinct intermediate lines were demonstrated in perivascular macrophages. Histochemical study showed these cells to contain free as well as esterified cholesterol. Gas chromatographic analysis of very long chain fatty acids (VLFA) from the demyelinated cerebral white matter showed a marked increase of C26:0 fatty acid in cholesterol esters and above-normal values for C24:0 and C24:1 in gangliosides. It is suggested that the condition was a heterozygote form of X-linked ALD. Patients with neurodegenerative symptoms with or without adrenal insufficiency can easily be screened for X-linked ALD by VLFA analysis in blood or cultured fibroblasts.

Neurodegenerative course in ceramidase deficiency (Farber disease) correlates with the residual lysosomal ceramide turnover in cultured living patient cells.

Farber's lipogranulomatosis is an inborn lipid storage disease characterized by tissue accumulation of ceramide due to deficient activity of lysosomal ceramidase. Symptoms include painful swelling of joints, subcutaneous nodules, a hoarse cry, hepatosplenomegaly and nervous system dysfunction of markedly variable degree. In most cases the neural dysfunction rather than the general dystrophy, seems to limit the duration of Farber disease. We examined whether the severity can be shown as a function of ceramide turnover by lysosomal ceramidase. The lysosomal degradation of sphingomyelin-derived ceramide was studied in situ in patient skin fibroblasts and lymphoid cells loaded with LDL-associated radioactive sphingomyelin. We could show for the first time a significant correlation between the ceramide accumulated in situ and the severity of Farber disease. Our method provides an alternative means for determining ceramide degradation by lysosomal ceramidase, but in intact cells. The relatively simple method is at least of the same diagnostic use for Farber disease as the in vitro assay of acid ceramidase using cell homogenates and may also have some prognostic use.

Progressive cerebellar ataxia, proximal neurogenic weakness and ocular motor disturbances: hexosaminidase A deficiency with late clinical onset in four siblings.

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.

Prenatal enzymic diagnosis in 24 pregnancies with risk of Krabbe disease .

In 24 pregnancies at risk for Krabbe disease (KD) monitored by amniocentesis in the 15th to 18th week, the amniocytic galactosyl ceramide beta-galactosidase activity was either lower than 11% (n = 12) or higher than 28% (n = 12) of the mean control amniocytic activity (n = 27). For the low activity group, prenatal KD diagnoses were made and the fetuses aborted. In the tissue material available from 11 of them the diagnoses were confirmed enzymatically. Three fetuses were studied electron microscopically and typical tubulo-spicular inclusions were found in the 'pre-globoid cells'. In the children already born from the group with more than 28% of amniocytic lipid beta-galactosidase activity no signs of KD were detectable. In two of the families at risk of KD, four pregnancies followed the monitored ones, but, for obscure reasons, it came to our knowledge only after the birth of these four children; enzymic studies revealed one KD patient and three unaffected children (two of them possibly heterozygous). When comparing the total sample of 12 + 1 KD cases and 12 + 3 unaffected with the theoretical 7:31 ratio for affected/unaffected offspring, a significant prevalence of affected (chi 2 = 6.86; alpha = 0.01) was found.

Enzymic diagnosis in 27 cases with Gaucher's disease.

A method is described for the assay of glucosyl ceramide beta-glucosidase (glucocerebrosidase) in white blood cells, cultured fibroblasts and amniotic cells, and in tissue homogenates. Glucosyl ceramide extracted from Gaucher spleen and labelled by catalytically adding tritium to the ceramide double bonds was used as the substrate in the presence of pure sodium cholate as detergent. The specificity of the test was established by demonstrating the enzyme deficiency in 25 cases with Gaucher's disease type 1 and 2. In two prenatal cases quantitative liver lipid analysis showed that glucosyl ceramide storage starts in Gaucher fetuses when they are about 20 weeks old.

Quantified increases of cholesterol, total lipid and globotriaosylceramide in filipin-positive Niemann-Pick type C fibroblasts.

BACKGROUND: Niemann-Pick disease type C (NPC) is a neurovisceral lysosomal lipidosis caused in most cases by mutations in the NPC1 gene that codes for the cholesterol regulating NPC1 protein. METHODS: Cultured skin fibroblasts from 11 NPC patients aged 0.25 to 34 years at diagnosis with different severity of neurologic and visceral involvement, diagnosed by the cytochemical filipin test for lysosomally stored cholesterol, were analyzed for lipid composition. Cholesterol and other lipids were separated on thin-layer chromatography from fibroblast total lipid extracts, quantified by densitometry and compared with the total cell lipid mass. RESULTS: Cholesterol concentration in the patient cells was 1.5 to 5-fold higher than normal and total lipids up to 2.4-fold normal. Cholesterol and total lipids were particularly high in cells from NPC patients aged less than about 6 years, and for the whole patient series the abundance of fibroblast cholesterol was correlated with the tentatively assessed clinical disease severity. The findings in NPC suggested that NPC1 protein has a role not only in the balance of cholesterol but also the distribution of the total cell lipid mass. Another increase found in the NPC cells was that of a minor lipid fraction, globotriaosylceramide (Gb3, known as a cell signalling glycolipid). Gb3, in the average of its very variable individual concentrations, was about 2.5-fold higher in the NPC cell group as compared to normal or pathologic control group, but there was no correlation of Gb3 with the other lipid concentrations studied. CONCLUSIONS: For NPC diagnosis, the fibroblast cholesterol and total lipid quantification can be used as an alternative to the usual filipin test for lysosomal cholesterol, but both test methods are prone to equivocal results in cells from a small fraction of atypical NPC patients, where chemical testing in organ biopsies or mutational analysis of the NPC1 gene should be tried.

Neuraminidase assay in cultured human fibroblasts: in situ versus in vitro procedures.

Further investigations have been carried out to characterize a published procedure of neuraminidase assay, in which the activity is measured directly on the cell culture layer. The pH optimum was 4.0. A Vmax value of 130 nmol/mg/h and a K(m) of 0.3 mmol/l were found. During incubation in the acid buffer, arylsulphatase activity was released into the medium, whereas neuraminidase activity remained attached to the cells. The in situ method allowed an unequivocal diagnosis of primary and secondary neuraminidase deficiencies. Because of its simplicity and reliability, the method appears useful as a routine method in clinical laboratories.

A simple method for screening for Farber disease on cultured skin fibroblasts.

Farber disease is an inborn lysosomal storage disorder characterized by accumulation of ceramide in the patient's tissues due to the deficient activity of acid ceramidase. Currently, confirmation of the diagnosis is performed in an extremely limited number of laboratories. We therefore developed a procedure which does not require any particular sphingolipid substrate and is based on the quantitation of ceramide levels in cultured skin fibroblasts. In the method we devised, the ceramide present in cellular lipid extracts subjected to mild alkaline hydrolysis was quantified using the commercially available diacylglycerol kinase kit. We show that both primary cultures of skin fibroblasts and SV40-transformed fibroblasts derived from a series of patients with Farber disease exhibit ceramide excess as compared to their normal counterparts (2345-17 153 pmol/mg cell protein in Farber cells vs. 432-1298 pmol/mg cell protein in controls). Use of this simple method should greatly facilitate the biochemical diagnosis of Farber disease.

Model SV40-transformed fibroblast lines for metabolic studies of human prosaposin and acid ceramidase deficiencies.

Skin fibroblasts from patients with Farber disease (acid ceramidase deficiency) and from two siblings of the only known family affected with prosaposin deficiency were transformed by transfection with a plasmid carrying the SV40 large T antigen. The prosaposin-deficient transformed cell lines conserved their original metabolic defects, and in particular they were free of detectable immunoreactivity when using anti-saposin B and anti-saposin C antisera. Ultrastructurally, the cells contained heterogeneous lysosomal storage products. As found for their parental cell lines, the SV40-transformed fibroblasts exhibited deficient in vitro activities of lysosomal ceramidase and beta-galactosylceramidase, but a normal activity of acid sphingomyelinase. As observed for SV40-transformed fibroblasts from Farber disease, degradation of radioactive glucosylceramide or low density lipoprotein-associated radiolabelled sphingomyelin by the prosaposin-deficient cells in situ showed a clear impairment in the turnover of lysosomal ceramide. Ceramide storage in prosaposin-deficient cells was also demonstrated by ceramide mass determination. In contrast to acid ceramidase deficient cells, both the accumulation of ceramide and the reduced in vitro activity of acid ceramidase in cells from prosaposin deficiency could be corrected by addition of purified saposin D. The data confirm that prosaposin is required for lysosomal ceramide degradation, but not for sphingomyelin turnover. The SV40-transformed fibroblasts will be useful for pathophysiological studies on human prosaposin deficiency.

Ultrastructural pathology of eccrine sweat gland epithelial cells in globoid cell leukodystrophy.

Three of four children were recognized by deficient beta-galactocerebrosidase activities as having globoid cell leukodystrophy inclusions in sweat gland epithelial cells, similar in ultrastructure to those seen in Schwann cells. This observation in globoid cell leukodystrophy emphasizes the need to include sweat gland epithelial cells in examinations of skin in globoid cell leukodystrophy, as well as in any neurometabolic disorder.

Late-onset globoid cell leukodystrophy: unusual ultrastructural pathology and subtotal beta-galactocerebrosidase deficiency.

An 11-year-old girl was found to have severely reduced beta-galactocerebrosidase activity as evidence of late-onset globoid cell leukodystrophy, while her mother had almost normal enzyme activity in circulating white blood cells. Clinically, the patient showed a remitting course marked by seizures, ataxia, white-matter disease on computed tomographic scan, and reduced conduction velocities of peripheral nerves. Symptoms improved somewhat around the age of 10 years. Two sural nerve biopsies, performed 6 years apart, disclosed a demyelinating neuropathy. By electron microscopy, membrane-bound vacuolar lysosomes in Schwann cells of myelinated axons, unlike the typical needlelike inclusions seen in classic infantile globoid cell leukodystrophy, were present in both specimens. Thus, clinical, morphologic, and biochemical data in this patient--and her mother--emphasize, compared with past reports on late-onset globoid cell leukodystrophy, considerable variation in the nosologic spectrum of late-onset globoid cell leukodystrophy and conspicuous differences from classic infantile globoid cell leukodystrophy.