Ikuo Goto

Lysosulfatide (Sulfogalactosylsphingosine) Accumulation in Tissues from Patients with Metachromatic Leukodystrophy

Abstract: We describe here a sensitive assay method for lysosulfatide (sulfogalactosylsphingosine) in human tissues using HPLC. The method involves extraction of lipids, saponification, isolation using a C18 Sep‐Pak column, derivatization with o‐phthalaldehyde, and detection of the fluorescent lysosulfatide using HPLC. In control subjects, a small amount of lysosulfatide was detected in the cerebral white matter (9–35 pmol/mg of protein), spinal cord (35 pmol/mg of protein), sciatic nerve (14 pmol/mg of protein), and kidney (∼2 pmol/ mg of protein) but not in the cerebral gray matter and liver. A marked accumulation of the lipid was noted in tissues from six patients with metachromatic leukodystrophy (MLD). The concentration of lysosulfatide was high in the cerebral white matter, spinal cord, and sciatic nerve (223–1,172 pmol/mg of protein). Even in the cerebral gray matter, kidney, and liver, where lysosulfatide was hardly detected in the control sample, a considerable amount (3–45 pmol/mg of protein) accumulated in MLD patients. The concentration and distribution pattern of lysosulfatide were similar to those of galactosylsphingosine (psychosine) accumulated in patients with Krabbe disease. Therefore, the accumulation of lysosulfatide may explain the demyelination in patients with MLD, as is the case with Krabbe disease.

The twitcher mouse: accumulation of galactosylsphingosine and pathology of the central nervous system.

In the twitcher mouse, a murine model of globoid cell leukodystrophy (GLD), pathological changes of various parts of the central nervous system correlated well with the concentration of galactosylsphingosine (psychosine). The development of GLD lesions was more obvious in tracts with a more rapid progression of myelination. It was suggested that accumulation of galactosylsphingosine subsequent to myelin maturation caused suicidal death of myelin forming cells.

Accumulation of lysosulfatide (sulfogalactosylsphingosine) in tissues of a boy with metachromatic leukodystrophy

Abnormal accumulation of lysosulfatide (sulfogalactosylsphingosine) was evident in autopsied tissues from a boy with late-infantile metachromatic leukodystrophy. The concentration was high in the cerebral white matter, spinal cord and sciatic nerve (116-787 pmol/mg protein) and low in the cerebral gray matter, kidney and liver (4-40 pmol/mg protein). As is the case with galactosylsphingosine, lysosulfatide inhibited cytochrome c oxidase activity, in a dose-dependent manner. Judging from the tissue distribution of the accumulated lysosulfatide and because of the cytotoxicity, the lysosulfatide presumably explains the demyelination seen in the nervous tissues of patients with metachromatic leukodystrophy.

Juvenile Sandhoff disease: a Japanese patient carrying a mutation identical to that found earlier in a Canadian patient

A 35-year-old Japanese man with juvenile Sandhoff disease is described. He showed progressive neurogenic muscular atrophy, cerebellar ataxia and mental deterioration, beginning at age 10 years. The accumulation of GM2 ganglioside in the submucosal nerve cell was confirmed by positive immunostaining using anti-GM2 ganglioside antibody. Biochemical evaluation revealed nearly absent beta-hexosaminidase A and B activities in leukocytes and cultured fibroblasts. Hydrolysis of [3H]globoside I in the intact fibroblasts was apparently disturbed but the rate of hydrolysis was higher than those seen in cells from patients with infantile Sandhoff disease. Analysis of the beta-hexosaminidase beta-subunit gene of the patient disclosed a point mutation (a G-to-A transition) within intron 12. The mutation generates a new splice junction resulting in a 24-base insertion between exons 12 and 13 in the processed mRNA and consequently an 8-amino acid insertion in the translation product. This mutation is identical to that originally found in a Canadian patient with juvenile Sandhoff disease. A possible relationship with the clinical phenotype and the gene abnormality is discussed.

Biosynthesis of galactosylsphingosine (psychosine) in the twitcher mouse.

In attempts to elucidate the origin of accumulated galactosylsphingosine in the twitcher mouse, a murine model of human globoid cell leukodystrophy (Krabbes disease), UDP-galactose: sphingosine galactosyltransferase activity was assayed in tissues from normal and twitcher mice. Among several tissues from normal, 20 day postnatal mice, the highest galactosyltransferase activity was found in the brainstem and spinal cord, followed by cerebrum, kidney and liver, in that order. Chronologically, the enzyme activity in the central nervous tissue increased with age, reached a maximum at 25 postnatal days, and declined thereafter. In the kidney and liver, however, the activity remained much the same during development. In the twitcher mouse, developmental change in the enzyme activity was similar to that seen in control mouse, but the decrease in activity in the central nervous tissue after the 25 postnatal days was more rapid. The galactosyltransferase activity and the accumulation of galactosylsphingosine in the tissue of the twitcher mouse were closely related; where and when the enzyme activity was higher, the greater was the accumulation of galactosylsphingosine in the tissue of the twitcher mouse. These results strongly suggest that the accumulated galactosylsphingosine in the twitcher mouse is synthesized mainly by UDP-galactose: sphingosine galactosyltransferase.

A sensitive assay of lysogangliosides using high-performance liquid chromatography.

Lysogangliosides, LGM1, LGM2 and LGM3, each carrying a single sphingoid base (i.e., C18:1, C18:0, C20:1, C20:0), were prepared and a sensitive assay method of these lipids using HPLC was developed. The method involves fluorescence derivatization of the free amino group of the molecule with o-phthalaldehyde, separation of the molecular species of each lysoganglioside using reversed-phase HPLC and assay on the basis of a known amount of one of the lysogangliosides, as the internal standard. Using this method, lysoganglioside can be accurately assayed in the range of 5-1000 pmol. For assay of the lipid in the tissue, crude isolation procedures including extraction of lipids, Folchs partition and DEAE-Sepharose and AG 1-X2 column chromatographies were required before the fluorescence derivatization. In the normal human and the bovine cerebral cortex, 0.4-2.0 pmol/mg protein of LGM1 containing C18:1 and C20:1 sphingosine residues were detected. In the frontal cortex from a patient with Sandhoff disease, an abnormal accumulation (55-78 pmol/mg protein) of LGM2 was noted. Among various molecular species, LGM2 containing C18:1 was the most abundant.

Decreased Fatty Acylation of Myelin Proteolipid Protein in the Twitcher Mouse

Abstract: 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 μM) inhibited only 17% of the acylation in vitro, it may be responsible for the reduced acylation of PLP in vivo.

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.

The twitcher mouse: attenuated processes of Schwann cells in unmyelinated fibers

Morphological alterations occurring in Schwann cells of unmyelinated fibers (unmyelinated Schwann cells) were investigated in the sciatic nerve of the twicher mouse, a murine model of human globoid cell leukodystrophy. After postnatal day 10, the number of Schwann cell-axon units gradually increased and the number of unmyelinated axons per unit progressively decreased in the twitcher mouse. However, the total number of unmyelinated axons showed no significant differences between twitcher and normal mice. Thus, these alterations of unmyelinated Schwann cells in the twitcher mouse suggest that attenuated branching of cellular processes develops at an early stage and progresses together with progression of demyelination in this mutant.

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.