Metabolism of exogenous galactosylceramide in the twitcher mouse brain

The in vivo metabolism of galactosylceramide (gal-cer) in normal mice and in twitcher mice, a model of human GLD, was examined following intracerebral administration of gal-cer containing [1-¹⁴C]stearic acid. In normal mice, gal-cer was hydrolyzed to ceramide within 6 hours and ceramide was hydrolyzed to sphingosine and fatty acid. Most of the released fatty acid was immediately incorporated into other lipids. About 75% of injected gal-cer was hydrolyzed 80 hours after the injection, while in the twitcher mouse, only 17% of gal-cer was hydrolyzed. These results show that degradation of gal-cer is impaired in the twitcher mouse brain, but contradict to the fact that there was no evidence of any accumulation of gal-cer in the brain. This discrepancy may be due to the different sorting routes of biosynthesized and exogenously-administered gal-cer in the mouse brain. Most of the biosynthesized gal-cer is incorporated into myelin, while the injected gal-cer is incorporated into lysosomes.     

Accumulation of Galactosylsphingosine (Psychosine) in the Twitcher Mouse: Determination by HPLC

We developed a sensitive and simple procedure for determination of galactosylsphingosine (psychosine), using HPLC. The method involved extraction of lipids, separation by cation-exchange and C18 reverse-phase columns, and derivatization with o-phthalaldehyde. The fluorescent galactosylsphingosine was detected by HPLC. The amount of galactosylsphingosine was accurately assayed by simultaneous determination of glucosylsphingosine, as the internal standard. The detection limit was 0.5 ng/assay tube, and the quantitative range of the method was up to 750 ng. This procedure was applied to tissue from the twitcher mouse, an animal model of human globoid cell leukodystrophy, as well as tissue from normal and carrier mice. In the latter mice, a small amount of galactosylsphingosine was detected in the spinal cord (21.6-37.2 ng/100 mg wet weight) but not in the cerebrum and sciatic nerve. Marked accumulation of galactosylsphingosine was noted in the nervous tissues of the twitcher strain, even on postnatal day 4. The concentration of galactosylsphingosine was greater in the peripheral than in central nervous tissues. The spinal cord and brainstem contained more galactosylsphingosine than did the cerebrum and cerebellum. The concentration increased with age from 764 ng/100 mg in the sciatic nerve at 4 days to 5,910 ng/100 mg at 37 days. These data correlate well with the pathological changes; tissues containing higher concentrations of galactosylsphingosine show earlier and more severe pathological changes than those containing lower concentrations, thereby indicating the close link of galactosylsphingosine to the pathogenesis of the twitcher mouse.     

Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse

In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous [³²P]ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using [³H]methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the [³H] methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.     

Glycosylceramide Synthesis in the Developing Spinal Cord and Kidney of the Twitcher Mouse, an Enzymatically Authentic Model of Human Krabbe Disease

Abstract: UDP-galactose:ceramide galactosyltransferase activity was assayed in the spinal cord and kidney of the recently discovered neurological mutant, the twitcher mouse, which is an enzymatically authentic model of human globoid cell leukodystrophy (Krabbe disease). The activity in the spinal cord was essentially normal during the early myelination period up to 15 days. There was a slight reduction at 20 days. At 25 and 33 days, the galactosyltransferase activity was drastically reduced compared to controls. In contrast, the galactosyltransferase activity in the kidney of twitcher mice remained normal throughout the developmental stages examined. Activity of the control enzyme UDP-glucose:ceramide glucosyltransferase was always normal in both the spinal cord and kidney. Thus, reduction of galactosylceramide synthesis occurs in the CNS secondarily to the pathological alteration of the oligodendroglia. No such reduction occurs in the kidney, at least for the last step of galactosylceramide synthesis. Reduced synthesis as the result of metabolic regulation in the presence of the catabolic block is therefore unlikely to be the cause of the lack of abnormal accumulation of galactosylceramide in the kidney of patients with globoid cell leukodystrophy.     

Novel Procedure for Measuring Psychosine Derivatives by an HPLC Method

We developed a sensitive and simple method to determine galactosylsphingosine and glucosylsphingosine as a 4-fluoro-7-nitrobenzofurazan autofluorescent compound, using HPLC equipped with a Showdex sugar column. Amounts of galactosylsphingosine were successfully measured in the picomole range. This novel procedure is more stable and simpler than the previous method using o-phthalaldehyde. It was applied to tissues from the twitcher mouse, an animal model of human globoid cell leukodystrophy. The amount of galactosylsphingosine was 34-102 micrograms/kg of wet tissues in control cerebrum and cerebellum, whereas in twitcher mice the range was 2,251-4,228 micrograms/kg of wet tissues. The psychosine concentration was also increased in the liver and kidney of twitcher mice, respectively, 1,513 micrograms and 1,106 micrograms/kg of wet tissue (normal liver, 125 micrograms; normal kidney, 74 micrograms/kg of wet tissue). This novel procedure is useful for the pathochemical evaluation of lysosphingolipids in various sphingolipidoses as well as in other neuropathological and cellular conditions.     

Galactosylceramidase deficiency causes sperm abnormalities in the mouse model of globoid cell leukodystrophy

The classical recessive mouse mutant, "the twitcher," is one of the several animal models of the human globoid cell leukodystrophy (Krabbe disease) caused by a deficiency in the gene encoding the lysosomal enzyme galactosylceramidase (GALC). The failure to hydrolyze galactosylceramide (gal-cer) and galactosylsphingosine (psychosine) leads to degeneration of oligodendrocytes and severe demyelination. Substrate for GALC is also the galactosyl-alkyl-acyl-glycerol (GalAAG), precursor of the seminolipid, the most abundant glycolipid in spermatozoa of mammals. In this paper, we report the pathobiology of the testis and sperm in the twitcher mouse and demonstrate the importance of GALC for normal sperm maturation and function. The GALC deficit results in accumulation of GalAAG in the testis of the twitcher mouse. Morphological studies revealed that affected spermatozoa have abnormally swollen acrosomes and angulation of the flagellum mainly at midpiece-principal piece junction. Multiple folding of the principal piece was also observed. Electron microscopy analysis showed that in the twitcher sperm, acrosomal membrane is redundant, detached from the nucleus and folded over. Disorganization and abnormal arrangements of the axoneme components were also detected. These results provide in vivo evidence that GALC plays a critical role in spermiogenesis.     

Establishment of a galactocerebrosidase-deficient twitcher mouse cell line that expresses galactocerebrosidase activity in hybrids with control human fibroblasts

Summary Primary cell cultures from twitcher (galactocerebrosidase deficient) mice were made by enzymatic dispersion and explantation of skin obtained from 3-d-old littermates of atwi+/twi×twi+/twi mating. Galactocerebrosidase activity remained deficient for two twitcher cell lines, TM-1 and TM-2, and both lines demonstrated an initial period of growth decline, followed by accelerated growth. The TM-2 line has been subcultured for more than 3.5 yr, has a modal chromosome number of 63, a doubling time of approximately 16 h, and has remained galactocerebrosidase deficient throughout its life span. These data indicate this to be an established twitcher cell line that can be continuously maintained in culture as a transformed galactocerebrosidase-deficient mouse cell line. This established line was rendered 6-thioguanine resistant so that the cells could be fused with control human fibroblasts and selected for hybrid lines in hypoxanthine-aminopterin-thymidine medium. Also, the established twitcher cells were crossed with neomycin-resistant control human fibroblasts and selected in G418 medium. Several of the hybrid lines from both crosses had higher than deficient levels of galactocerebrosidase activity initially, followed by a decrease to twitcher levels during subculture, whereas other lines retained high levels of activity. These results indicate that twitcher-human somatic cell hybrids will express galactocerebrosidase activity and thus may be useful for determining the human chromosome or chromosomes associated with this expression. Partial support for these studies was provided by a National Institutes of Health AREA grant (HD21222-01) and a NIH subcontract to Clark University from the Shriver Center for Mental Retardation This research forms a portion of studies performed to fulfill the requirements for the Ph.D. degree in biology at Clark University for J. T. K.     

Quantification of galactosylsphingosine in the twitcher mouse using electrospray ionization-tandem mass spectrometry.

Globoid cell leukodystrophy (Krabbe disease) is an autosomal recessive inherited neurodegenerative disorder caused by the deficiency of the lysosomal enzyme beta-galactosylceramidase. The pathogenesis of the disorder has been proposed to arise from the accumulation of the cytotoxic metabolite galactosylsphingosine (psychosine). The twitcher mouse is a naturally occurring murine model of globoid cell leukodystrophy. We have developed a rapid, sensitive, and specific mass spectrometric method for determining the galactosylsphingosine concentration in the tissues of twitcher mice. Galactosylsphingosine is extracted from the tissues in methanol, isolated using strong cation-exchange and C18 solid-phase extraction chromatography, and then directly analyzed using electrospray ionization-tandem mass spectrometry. A lactosylsphingosine internal standard has been employed for quantification. The assay demonstrated significant accumulation of galactosylsphingosine in the brain, spinal cord, and kidney of twitcher mice. It is anticipated that this method may be of use in the monitoring of experimental therapies for globoid cell leukodystrophy.     

Decreased Fatty Acylation of Myelin Proteolipid Protein in the Twitcher Mouse

We examined chronological changes of myelin proteins of the brainstem and spinal cord of the twitcher mouse (15, 20, and 30 days old), a murine model of human globoid cell leukodystrophy caused by a genetic deficiency of galactosylceramidase I activity. The yield of myelin was normal until postnatal day 20, whereas galactosylsphingosine (psychosine) accumulated with age in myelin. The protein profiles of myelin and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase in the myelin remained normal throughout the experimental period. Fatty acylation of proteolipid protein (PLP) was examined in a cell-free system by incubation of myelin with [3H]palmitic acid, CoA, and ATP, and was normal at postnatal day 15, but decreased after postnatal day 20. Decreased fatty acylation of PLP was also observed in the twitcher mouse at postnatal day 20 when the isolated myelin was incubated with [14C]palmitoyl-CoA in the absence of ATP and CoA, or the slices of brainstem and spinal cord were incubated with [3H]palmitic acid. The activity of fatty acid:CoA ligase was reduced in myelin. These data suggest that decreased acylation of PLP in twitcher mouse myelin is probably due to reduced activities for both activation and transfer of fatty acid into PLP and that metabolic disturbance is present in myelin because acylation of PLP has been shown to occur in myelin membrane. Although psychosine (200 microM) inhibited only 17% of the acylation in vitro, it may be responsible for the reduced acylation of PLP in vivo.     

Cellular uptake and lysosomal delivery of galactocerebrosidase tagged with the HIV Tat protein transduction domain

A number of studies have shown that a short peptide, the protein transduction domain (PTD) derived from the HIV-1 Tat protein (Tat-PTD) improved cellular uptake in vitro and distribution in vivo of recombinant proteins bearing such PTDs when administered systemically. To investigate the effects of Tat-PTD addition on the subcellular localization of the lysosomal enzyme galactocerebrosidase (GALC, EC 3.2.2.46) and with a view towards designing improved therapeutic strategies for Krabbe disease (globoid cell leukodystrophy), mouse GALC was tagged C-terminally with the Tat-PTD. Compared with unmodified GALC, GALC bearing a Tat-PTD, a myc epitope and 6 consecutive His residues [GALC-TMH (Tat-PTD, a myc epitope and 6 consecutive His residues)] was found to be secreted more efficiently. Also, GALC-TMH was found to be taken up by cells both via mannose-6-phosphate receptor (M6PR)-mediated endocytosis as well as by M6PR-independent mechanisms. GALC-TMH displayed increased M6PR-independent uptake in fibroblasts derived from twitcher mice (a murine model of globoid cell leukodystrophy) and in neurons derived from the mouse brain cortex compared with GALC lacking a Tat-PTD. Immunocytochemical analyses revealed that Tat-modified GALC protein co-localized in part with the lysosome-associated membrane protein-1. Complete correction of galactosylceramide accumulation was achieved in twitcher mouse fibroblasts lacking GALC activity following addition of GALC-TMH. Therefore, GALC-TMH not only maintained the features of the native GALC protein including enzymatic function, intracellular transport and location, but also displayed more efficient cellular uptake.     

Cerebroside and Sulfatide Biosynthesis in the Brain of Snell Dwarf Mouse: Effects of Thyroxine and Growth Hormone in the Early Postnatal Period

Snell dwarf mice (dw/dw) and normal mice (+/?) were injected with thyroxine (T₄) (1 μg/animal, four injections) and growth hormone (GH) (20 μg/animal, four injections) from the 5th to the 15th day of life. In the untreated dw/dw mouse brain, the specific activities of UDP-galactose:ceramide galactosyltransferase (CGalT), PAPS:cerebroside sulfotransferase (CST), and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) were decreased by 28, 25, and 37%, respectively, compared with the control untreated +/? mice. The major effect of T₄ was an increase of the brain CNP in the +/? mice (+40%) and dw/dw mice (+111%). The treatment with T₄ also brought to normal the level of CGalT in dw/dw brain; a somewhat less marked effect on CST was observed. The treatment with GH had a great stimulatory effect on CNP: the specific activity of this enzyme increased by 40 and 69% in +/? and dw/dw mouse brain, respectively. On the contrary, no effect of GH on the CGalT activity was observe in this study. Our results suggest that T₄ and GH may have both independent and complementary actions on the myelin-associated enzymes during the early postnatal period of brain development.     

Drastically Abnormal Gluco- and Galactosylceramide Composition Does Not Affect Ganglioside Metabolism in the Brain of Mice Deficient in Galactosylceramide Synthase

Mice that are genetically deficient in UDP-galactose: ceramide galactosyltransferase are unable to synthesize galactosylceramide. Consequently, sulfatide, which can be synthesized only by sulfation of galactosylceramide, is also totally absent in affected mouse brain. -Hydroxy fatty acid-containing glucosylceramide partially replaces the missing galactosylceramide. A substantial proportion of sphingomyelin, which normally contains only non-hydroxy fatty acids, also contains -hydroxy fatty acids. These findings indicate that -hydroxy fatty acid-containing ceramide normally present only in galactosylceramide and sulfatide is diverted to other compounds because they cannot be synthesized into galactosylceramide due to the lack of the galactosyltransferase. We have examined brain gangliosides in order to determine if -hydroxy fatty acid-containing glucosylceramide present in an abnormally high concentration is also incorporated into gangliosides. The brain ganglioside composition, however, is entirely normal in both the total amount and molecular distribution in these mice. One feasible explanation is that UDP-galactose: glucosylceramide galactosyltransferase does not recognize -hydroxy fatty acid-containing glucosylceramide as acceptor. This analytical finding is consistent with the relative sparing of gray matter in the affected mice and provides an insight into sphingolipid metabolism in the mouse brain.     

Psychosine Is as Potent an Inducer of Cell Death as C6-Ceramide in Cultured Fibroblasts and in MOCH-1 Cells

Cytotoxic capacity of psychosine (galactosylsphingosine) was evaluated in comparison with C6-ceramide in cultured fibroblasts and the glia-derived MOCH-1 cells that have characteristics of myelinating cells (1). Psychosine caused cytotoxic cell death and DNA fragmentation at concentrations similar to C6-ceramide and MOCH-1 cells were substantially more sensitive to their cytotoxic effects than fibroblasts. In this system, pretreatment with GM1-ganglioside failed to protect the cells from the deleterious effects of these compounds. These findings are consistent with the hypothesis that psychosine is the cytotoxic metabolite that causes apoptotic death of the oligodendrocyte in globoid cell leukodystrophy (Krabbe disease). They further suggest that the protective capacity of GM1-ganglioside is unlikely to be the explanation for the paradoxical improvement of the phenotype of globoid cell leukodystrophy in the mouse simultaneously deficient in two lysosomal -galactosidases, galactosylceramidase and GM1-ganglioside -galactosidase.     

Psychosine-induced alterations in peroxisomes of twitcher mouse liver

Krabbe disease is a neuroinflammatory disorder in which galactosylsphingosine (psychosine) accumulates in nervous tissue. To gain insight into whether the psychosine-induced effects in nervous tissue extend to peripheral organs, we investigated the expression of cytokines and their effects on peroxisomal structure/functions in twitcher mouse liver (animal model of Krabbe disease). Immunofluorescence analysis demonstrated TNF-α and IL-6 expression, which was confirmed by mRNAs quantitation. Despite the presence of TNF-α, lipidomic analysis did not indicate a significant decrease in sphingomyelin or an increase in ceramide fractions. Ultrastructural analysis of catalase-dependent staining of liver sections showed reduced reactivity without significant changes in peroxisomal contents. This observation was confirmed by assaying catalase activity and quantitation of its mRNA, both of which were found significantly decreased in twitcher mouse liver. Western blot analysis demonstrated a generalized reduction of peroxisomal matrix and membrane proteins. These observations indicate that twitcher mouse pathobiology extends to the liver, where psychosine-induced TNF-α and IL-6 compromise peroxisomal structure and functions.     

Saposins (Sphingolipid Activator Proteins) in the Twitcher Mutant Mouse

The twitcher mutant mouse, the animal model of Krabbe disease (human globoid cell leukodystrophy), is characterized by apparent deficiency of galactosylceramide beta-galactosidase activity. Saposin A and C, the heat-stable small sphingolipid activator glycoproteins, stimulate the activity of galactosylceramide beta-galactosidase as well as glucosylceramide beta-glucoside. The role of these saposins in the twitcher mutation was investigated. Boiled supernatant fractions, which contained saposins, were prepared from homogenates of twitcher brain, liver, kidney, and spleen. These preparations showed an almost identical effect on the activity of purified glucosylceramide beta-glucosidase (measured by hydrolysis of 4-methylumbelliferyl-beta-glucoside) with similar preparations from control tissues. The effect on the activity of galactosylceramide beta-galactosidase as well as 4-methylumbelliferyl-beta-glucoside beta-glucosidase in the twitcher brain and liver homogenates by authentic saposin A and C was similar to that in control tissues. These results suggest that the twitcher mutation does not affect the concentrations of saposin A or C or their interaction with galactosylceramide beta-galactosidase.     

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.     

Abnormal accumulation of galactosylceramide in the kidney of twitcher mouse

The kidney tissue of the twitcher mice, a neurological mutant caused by a genetic deficiency of galactosylceramidase, contains enormously increased amounts, up to 50 times normal, of galactosylceramide. The finding is in sharp contrast with those in the enzymatically equivalent human disease, globoid cell leukodystrophy (Krabbe disease), in which no specific abnormal accumulation of galactosylceramide occurs despite the same genetic block in the catabolic pathway. This indicates that the same genetic defect can result in entirely different consequences in different species. Caution must be exercised even when "authentic animal models" are utilized for studies of human diseases.     

T-cell dependent collagenous encapsulating response in the mouse liver to Mesocestoides corti (Cestoda)

Pollacco S., Nicholas W.L., Mitchell G.F. and Chaicharn Stewart A. 1978. T-cell dependent collagenous encapsulating response in the mouse liver to Mesocestoides corti (Cestoda). International Journal for Parasitology8: 457–462. Experiments with genetically hypothymic mice show that the tetrathyridial larvae of Mesocestoides corti (Cestoda) multiply much more rapidly in the liver than in normal mice. In the hypothymic mouse, collagen fibres are not laid down and the parasite is not encapsulated as it is in the normal mouse. Encapsulation probably restricts the parasite's multiplication, and it is suggested that the failure to encapsulate the parasite accounts for its more rapid multiplication in the hypothymic mouse. Fibrogenesis and encapsulation is restored to hypothymic mice by transferring syngeneic thymus cells, spleen cells or peritoneal exudate cells. It is concluded that the encapsulation of M. corti is a T-cell dependent process.