Genetic galactocerebrosidase deficiency (globoid cell leukodystrophy, Krabbe disease) in rhesus monkeys (Macaca mulatta)

Globoid cell leukodystrophy, or Krabbe disease, is a severe disorder of the peripheral and central nervous system myelin caused by deficient galactocerebrosidase (GALC) activity. This autosomal recessive disease affects humans and animals including dogs, mice, and rhesus monkeys. Cloning of the human and animal GALC genes opened opportunities for therapeutic trials using animal models. We describe the clinical, pathologic, and biochemical features of the affected rhesus monkey. Affected monkeys had very low GALC activity and a two base pair deletion in both copies of the GALC gene. Clinical signs of tremors, hypertonia, and incoordination led to humane euthanasia by 5 months of age. At necropsy, peripheral nerves were enlarged. Microscopically, the cerebral, cerebellar, and spinal cord white matter was infiltrated with periodic acid-Schiff-positive multinucleated globoid cells, and there was a striking lack of myelin. Peripheral nerve fibers were decreased in number and separated by Alcian blue- and safranin O-positive material. Myelin sheaths were greatly diminished. Lipid analysis of brains of 12-day-old and 158-day-old affected monkeys revealed a great excess of psychosine in white matter. The rhesus monkey model will be especially useful for exploring treatment options, including prenatal bone marrow transplantation and various approaches to gene therapy.     

Krabbe's disease: globoid cell leukodystrophy

The clinical features of regression in mental and motor development of a 7-month-old child are reported, together with the demonstration of a profound deficiency of galactosylceramide beta-D-galactosidase in a liver biopsy. The diagnosis of Krabbe''s disease or globoid cell leukodystrophy (GLD) is therefore unequivocally established. The clinical features and morbid anatomical findings permitting the diagnosis of GLD in two of the child''s sibs are summarized. This is the first report from Newfoundland of this inborn error of sphingolipid metabolism.     

Prenatal diagnosis of globoid cell leukodystrophy (Krabbe's disease)

Summary A case of globoid cell leukodystrophy (Krabbe's disease) was diagnosed prenatally by demonstrating a profound deficiency of cerebroside -galactosidase in cultured amniotic cells. The diagnosis was confirmed in the fetus aborted in the 19th week. In the cell-free amniotic fluid, normal enzyme activity was found. This finding, which had been demonstrated in a previous case, is discussed.     

An accumulation of galactocerebroside in kidney from mouse globoid cell leukodystrophy (twitcher)

The twitcher mouse is genetically determined mutant characterized by a deficiency of galactocerebroside beta-galactosidase. In this study, a significant accumulation of galactocerebroside was demonstrated in twitcher mouse kidney. The data suggest that mouse Krabbe's disease is not only involved in CNS, but also in visceral organs.     

Prenatal enzymatic diagnosis of Krabbe disease (globoid-cell leukodystrophy) using chorionic villi

Summary Sixteen pregnancies in families with children enzymatically diagnosed as having Krabbe disease (KD) were monitored for prenatal KD using the assay of galactosyl ceramide -galactosidase (GCG) in uncultured chorionic villi (CV), cultured CV, or cultured amniotic fluid cells (AFC). Prenatal KD diagnoses were made for 5 pregnancies on the basis of lower than 10% normal GCG activity in cultured CV or AFC. Uncultured CV were studied in 3 out of the 5 KD embryos, although the GCG activities of 14%–23% as compared with control villi were diagnostically inconclusive; the relatively high activities were considered to be caused by maternal GCG contamination of these very small villus samples. Although the villi from 6 of the other pregnancies yielded more conclusive results, the use of uncultured CV alone is not recommended for prenatal KD diagnosis, this material being subject to possible uncontrolled contamination with maternal enzyme.     

Galactosylceramide- and lactosylceramide-loading studies in cultured fibroblasts from normal individuals and patients with globoid cell leukodystrophy (Krabbe's disease) and GM1-gangliosidosis

The metabolism of galactosylceramide and lactosylceramide in cultured fibroblasts was studied using the lipid-loading test. These compounds were incorporated into the fibroblasts yet only small amounts of the incorporated lipids were hydrolyzed unless additional phospholipid was mixed with the glycolipid before loading. Among phospholipids, phosphatidylserine was the most effective for incorporation and hydrolysis of the glycolipids, while phosphatidylcholine inhibited the incorporation of the glycolipids. Using filtration techniques, light scattering analyses and subcellular fractionation, the particle size of glycolipid in the culture medium was found to be critically important for the incorporation of the lipids into the cells and their transportation to the lysosomes. The particle sizes of the glycolipids were decreased by mixing with phosphatidylserine. Furthermore, the negative charge in phosphatidylserine may be necessary for the glycolipid transportation into the lysosomes. In fibroblasts from patients with globoid cell leukodystrophy, 40-50% of galactosylceramide was hydrolyzed on the 4th day of culture, a time when the control fibroblasts had hydrolyzed it about 80%. This finding is in contrast with observations made on fibroblasts with other sphingolipidoses which showed near-zero degradation in corresponding substrate-loading tests. In fibroblasts from patients with either globoid cell leukodystrophy of GM1-gangliosidosis, hydrolysis of lactosylceramide was fairly normal yet somewhat lower than control values on any day of culture, thereby indicating that, in the loading tests, lactosylceramide seems to be hydrolyzed with similar levels of enzyme activities by two distinct beta-galactosidases, galactosylceramidase and GM1-ganglioside beta-galactosidase.     

Enzyme replacement with liposomes containing beta-galactosidase from Charonia lumpas in murine globoid cell leukodystrophy (twitcher)

Enzyme replacement with liposomes containing beta-galactosidase obtained from charonia lumpas was carried out in murine globoid cell leukodystrophy (GLD). Charonia lumpas beta-galactosidase was able to hydrolyze galactocerebroside trapped into liposomes prepared from lecithin, cholesterol and sulfatide (molar ratio; 7:2:1). Liposomes containing charonia lumpas beta-galactosidase were successfully incorporated into the mouse tissues. 3H-galactocerebroside labeled liposomes were also incorporated into mouse liver, spleen and other tissues. The accumulation rate of 3H-galactocerebroside into twithcer mice liver and spleen was almost 40 to 100 times higher than those of controls and degraded to 70 to 80% of accumulated radioactivity of 3H-galactocerebroside by single injection of liposomes containing charonia lumpas beta-galactosidase. Results suggest that exogeneous enzyme trapped in liposomes can be useful for the correction of accumulated compound.     

Sphingolipid profile in the CNS of the twitcher (globoid cell leukodystrophy) mouse: a lipidomics approach.

Globoid cell leukodystrophy (Krabbe disease) is caused by mutations in galactosylceramidase, a lysosomal enzyme that acts to digest galactosylceramide, a glycolipid concentrated in myelin, and psychosine (galactosylsphingosine). Globoid cell leukodystrophy has been identified in many species including humans and twitcher mice. Several studies on human tissue have examined the lipid profile in this disease by gas, liquid or thin layer chromatography. Electrospray ionization tandem mass spectrometry combined with reverse phase HPLC has become a powerful alternative strategy, used here to compare the sphingolipid profile of pons/medulla tissue from twitcher mice with control tissue. In this lipidomics LC-MS approach, we scanned for precursors of m/z 264 to obtain a semi-quantitative profile of ceramides and galactosylceramides. Sphingosine-1-phosphate, C18:0 ceramide, C22:0 ceramide and C24:0 ceramide levels were reduced in the pons/medulla of twitcher mice compared to levels in control mice at 31 and 35-37 days of age. The levels of C22:0 and C24:0 galactosylceramide were similar between twitcher and control specimens and there was a trend toward reduced levels of C24:1 galactosylceramide and C24:1 hydroxy-galactosylceramide in twitcher specimens. Psychosine, C 16:0 ceramide and C 18:0 galactosylceramide levels were increased in the CNS of twitcher mice compared to levels in control mice. These data indicate that there is a trend toward decreased levels of long chain fatty acids and increased levels of shorter chain fatty acids in galactosylceramides and ceramides from twitcher mice compared with control mice, and such changes may be due to demyelination characteristic of acute pathology.     

Globoid cell leukodystrophy is a generalized galactosylsphingosine (psychosine) storage disease

Galactosylsphingosine (psychosine) in somatic organs from a patient with globoid cell leukodystrophy and from the twitcher mouse, an animal model of human globoid cell leukodystrophy was assayed. There was an abnormal accumulation of galactosylsphingosine as in nervous tissues, albeit the concentrations being lower than those in nervous tissues. Galactosylsphingosine accumulation in the kidney of the twitcher mouse increased with age. These findings indicate that globoid cell leukodystrophy is a generalized galactosylsphingosine storage disease.     

Globoid cell leukodystrophy (Krabbe's disease): isolation of myelin with normal glycolipid composition

Myelin was isolated from the brain of a patient with Krabbe's globoid cell leukodystrophy at 0.4% of the normal yield. Despite the exceedingly low yield, the fraction appeared morphologically clean, and consisted mostly of well-preserved myelin lamellae and few contaminating structures. Total lipid and cholesterol were slightly lower than in normal myelin. Total phospholipid was normal, but the ratio of ethanolamine phospholipid to lecithin was reversed. Total galactolipid was normal, and consisted only of cerebroside and sulfatide in normal proportions. The only sugar in cerebroside and sulfatide was galactose. The fatty acid composition of cerebroside and sulfatide was essentially normal with no deficiency of long-chain fatty acids and only with a reversed ratio of C(24:0) to C(24:1) in cerebroside. These data appear to exclude the previous postulate that abnormally rapid breakdown of myelin occurs in this disorder as the result of the formation of chemically abnormal myelin, deficient in sulfatide.     

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.     

Localization of the Krabbe disease gene (GALC) on chromosome 14 by multipoint linkage analysis.

The gene responsible for Krabbe disease, an autosomal recessive disorder caused by deficiency of galactocerebrosidase (GALC), was localized by multipoint linkage analysis on chromosome 14. Eight mapped dinucleotide repeat polymorphisms were tested for linkage to GALC. Two-point linkage analysis demonstrated close linkage of GALC and D14S48, with Z = 13.69 at theta = 0. Multipoint analysis yielded strong support for this finding, with maximum likelihood for GALC located within 1 cM of D14S48. This analysis also identified markers that clearly flank the GALC locus, as the map order of D14S53-GALC-D14S45 is favored by odds greater than 10(6):1. Additional support for close linkage of GALC and D14S48 comes from the apparent linkage disequilibrium between these two loci in a consanguineous Druze community in Israel. These data localize GALC to 14q24.3-q32.1.     

Mucopolysaccharidosis type II (Hunter disease): 13 gene mutations in 52 Japanese patients and carrier detection in four families

Southern blot analysis of the iduronate sulfatase (IDS) gene in 52 unrelated Japanese patients with mucopolysaccharidosis type II was carried out using a cDNA probe, and mutations in 13 patients (25%) were identified. Of these, 3 had partial gene deletions (in 2 the normal 9.4-kb fragment was absent and in 1 the normal 7.4-kb fragment was absent, as determined by Southern blot analysis using EcoRI-digested DNA, respectively), 2 had gene insertions (in 1 there was a unique 11.2kb fragment and in the other there was a unique 5kb fragment, determined by Southern blot analysis using EcoRI digested DNA), and 8 had rearrangements (in 6 the normal 9.4kb and 7.0kb fragments were absent and a unique 11.2kb fragment was present; in the remaining 2 patients there were different rearrangements). In these 13 patients, the similar Southern blot patterns were indicative of structural alterations of the IDS gene, as revealed when their DNA was digested with HindIII or PstI and probed with IDS cDNA. All patients with these structural alterations were in a clinically severe state, except for 1 with an intermediate clinical phenotype. Our analyses of four families among those of the 13 patients revealed that all four mothers were carriers. The detection of structural abnormalities led to a precise identification of Hunter heterozygotes and revealed one de novo rearrangement in a germ cell of one of the maternal grandparents.     

Krabbe disease: psychosine-mediated activation of phospholipase A2 in oligodendrocyte cell death

Globoid cell leukodystrophy (Krabbe disease) is an inherited neurological disorder caused by the pathogenomic accumulation of psychosine (galactosylsphingosine), a substrate for the deficient enzyme galactocerebroside beta-galactosidase. This study underscores the mechanism of action of psychosine in the regulation of oligodendrocyte cell death via the generation of lysophosphatidylcholine (LPC) and arachidonic acid (AA) by the activation of secretory phospholipase A2 (sPLA2). There was a significant increase in the level of LPC, indicating a phospholipase A2 (PLA2)-dependent pathobiology, in the brains of Krabbe disease patients and those of twitcher mice, an animal model of Krabbe disease. In vitro studies of the treatment of primary oligodendrocytes and the oligodendrocyte MO3.13 cell line with psychosine also showed the generation of LPC and the release of AA in a dose- and time-dependent manner, indicating psychosine-induced activation of PLA2. Studies with various pharmacological inhibitors of cytosolic phospholipase A2 and sPLA2 and psychosine-mediated induction of sPLA2 enzymatic activity in media supernatant suggest that psychosine-induced release of AA and generation of LPC is mainly contributed by sPLA2. An inhibitor of sPLA2, 7,7-dimethyl eicosadienoic acid, completely attenuated the psychosine-mediated accumulation of LPC levels, release of AA, and generation of reactive oxygen species, and blocked oligodendroyte cell death, as evident from cell survival, DNA fragmentation, and caspase 3 activity assays. This study documents for the first time that psychosine-induced cell death is mediated via the sPLA2 signaling pathway and that inhibitors of sPLA2 may hold a therapeutic potential for protection against oligodendrocyte cell death and resulting demyelination in Krabbe disease.