J. Nishimoto

Molecular cloning and expression of cDNA for murine galactocerebrosidase and mutation analysis of the twitcher mouse, a model of Krabbe's disease

Abstract: The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5′ end. It has a 2,278‐nucleotide sequence including a 2,004‐nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG → TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6‐kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3′‐untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.

A case of Hallervorden-Spatz disease: Progressive and intractable dystonia controlled by bilateral thalamotomy

We present a 10-year-old girl with Hallervorden-Spatz disease diagnosed clinically from the neurological manifestations and the characteristic MRI findings. Her main symptom, dystonia, was progressive and resistant to medication, but this dystonia was controlled by bilateral thalamotomy. No clinical progression of the symptoms was recognized at 21 months from the last operation.

A family with pseudodeficiency of acid α‐glucosidase

A unique family is presented which consists of a patient with the juvenile muscular dystrophy form of glycogenosis type II and four healthy individuals, both parents and sisters, with low acid ***α‐glucosidase activity. It was almost impossible to distinguish the homozygote from the heterozygous members by lymphocyte assay alone. In cultured skin fibroblasts, acid α‐glucosidase activity measured with a synthetic substrate was less than 1% of the normal mean value in the patient and about 15% in the parents. The activity toward glycogen was not detectable in the patient and was about 30% of the normal mean value in the parents. These values are also lower than expected in heterozygotes. To explain these results properly, a new mutant allele of acid α‐glucosidase is proposed. Both parents could be compound heterozygotes for the pseudodeficiency allele and the juvenile form of glycogenosis type II allele.

A case of pigmentary type of orthochromatic leukodystrophy with early onset and globoid cells

SummaryWe report herein a sporadic case of the pigmentary type of orthochromatic leukodystrophy with early onset and very rapid clinical course. The patients development was normal until 2 years old, when he experienced visual disturbance. Rapid deterioration resulted in death 1.5 years after the onset. Metachromatic leukodystrophy, globoid cell leukodystrophy and adrenoleukodystrophy were excluded by biochemical assays. Autopsy findings were compatible with the diagnosis of the pigmentary type of orthochromatic leukodystrophy. However, there were unique findings of severe neuronal loss and the collection of globoid-like cells in the interface of the gray matter and the white matter. Immunohistochemical staining of myelin basic protein, proteolipid protein and galactocerebroside demonstrated that these myelin constituents were equally preserved in the posterior column, while absent in the lateral and anterior columns of the spinal cord.

Study of pathogenesis in twitcher mouse, an enzymatically authentic model of Krabbe's disease.

The twitcher mouse was investigated by examining in vivo synthesis of galactosylceramide (Galcer) and galactosylsphingosine (Galsph) in a sciatic nerve culture, and in vitro enzymic activities for synthesis of Galcer and Galsph in the spinal cord from normal and affected mice. For the in vivo study, the sciatic nerve was incubated for 24 h in medium containing [3H]galactose, or [3H]-sphingosine-labeled Galcer or Galsph. With [3H]galactose, reduced synthesis of Galcer was found as early as 1 week of age and synthesis decreased to about 15% of normal value at 4 weeks. Increased Galsph was detected after 7 days of feeding with galactose. In a study of [3H]sphingosine-labeled Galcer and Galsph feeding, Galcer did not induce Galsph synthesis in either normal or affected mice, and synthesis of Galcer from Galsph was found only in normal mice, suggesting that Galcer was synthesized from sphingosine after hydrolysis of Galsph. In vitro, the activities of UDP-galactose: ceramide galactosyltransferase and UDP-galactose: sphingosine galactosyltransferase were reduced to less than 50% of control after 2 weeks of age in affected mice. We conclude that (1) decreased Galcer was due to impaired synthesis of Galcer, (2) Galsph was synthesized from galactose and not from deacylation of Galcer, and (3) Galsph accumulation was due not to increased synthesis but to decreased hydrolysis.

Metabolism of Cerebroside Sulphate and Subcellular Distribution of its Metabolites in Cultured Skin Fibroblasts Derived from Controls, Metachromatic Leukodystrophy, Globoid Cell Leukodystrophy and Farber Disease

The sphingolipidoses are generally understood to be lysosomal lipid storage diseases due to genetic enzymic defects of sphingolipid metabolism. However, the molecular mechanisms that lead to the clinical and pathological manifestations rernain largely obscure. Recent morphological and biochemical studies in neuronal storage diseases (Purpura and Suzuki, 1976) and animal models of gangliosidoses (Wood et al., 1985) suggested that altered membrane structure could be one of the causes of neuronal dysfunction. To understand the mechanism causing neurological dysfunctions in sphingolipidoses, clarification of the synthesis, translocation and insertion of sphingolipids in the different subcellular components in normal and pathological states is essential. This paper reports the metabolism of cerebroside sulphate (CS) and the subcellular distribution of its metabolite in cultured skin fibroblasts from normal control, metachromatic leukodystrophy (MLD; McKusick 25010), globoid cell leukodystrophy (GLD; McKusick 24520) and Farber disease (McKusick 22800).

Molecular form and subcellular distribution of acid β-galactosidase in fibroblasts from patients with GM1 gangliosidosis, Morquio B disease and galactosialidosis

The molecular form and subcellular distribution of acid beta-galactosidase in cultured fibroblasts from patients with beta-galactosidase deficiency (GM1-gangliosidosis, Morquio B disease and galactosialidosis) were studied, using antibodies against three different forms of the human enzyme: a high-molecular-weight multienzymic complex, a recombinant 84-kDa precursor, and a 64-kDa tryptic product of the precursor. The mature enzyme from normal fibroblasts was immunoprecipitated by the anti-complex and anti-64-kDa protein antibodies, but not by the anti-84-kDa precursor one. immunofluorescence staining of normal fibroblasts revealed the granular (lysosomal) distribution with anti-64-kDa protein antibody and the perinuclear reticular distribution with anti-84-kDa precursor antibody, probably representing the Golgi apparatus. Both patterns were demonstrated in Morquio B disease, but the residual enzyme activity was exclusively due to the mature enzyme. In Type 1 galactosialidosis, most of the expressed enzyme was detected as the precursor form with a perinuclear reticular distribution. In type 2 galactosialidosis, more than half of the enzyme activity was due to the mature form with a lysosomal distribution. Fibroblasts from a patient with GM1 gangliosidosis, expressing no beta-galactosidase mRNA, did not react against either anti-64-kDa protein antibody or anti-84-kDa precursor antibody. The combined use of immunoprecipitation and immunostaining was useful for analysing the pathophysiology of the intracellular processing and transport of the mutant beta-galactosidase.

Immunohistochemical Demonstration of GM2-Ganglioside in the Central Nervous System of a 19-week-old Fetus of Tay-Sachs Disease

Tay-Sachs disease (TSD, McKusick 27275) is an inherited neurodegenerative disorder due to the deficiency of β-hexosaminidase A (EC 3.2.1.52), which causes the accumulation of GM2-ganglioside (GM2) in the central nervous system (CNS) of the patients (O’Brien, 1983), but it remains unknown exactly when and where GM2 begins to accumulate in the affected tissues. We have studied its distribution in the CNS of a TSD fetus and an age-matched control by an immunohistochemical method in conjunction with the distribution of sphingolipid activator protein (SAP)-1, myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP).

Immunohistochemical Demonstration of GM2

Tay-Sachs disease (TSD, McKusick 27275) is an inherited neurodegenerative disorder due to the deficiency of β-hexosaminidase A (EC 3.2.1.52), which causes the accumulation of GM2-ganglioside (GM2) in the central nervous system (CNS) of the patients (O’Brien, 1983), but it remains unknown exactly when and where GM2 begins to accumulate in the affected tissues. We have studied its distribution in the CNS of a TSD fetus and an age-matched control by an immunohistochemical method in conjunction with the distribution of sphingolipid activator protein (SAP)-1, myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP).

Biochemical Heterogeneity in I-Cell Disease

Since we observed the normalization of intracellular hydrolases in some cell lines of I-cell disease (ICD) by 88 mmol/l sucrose, we have hypothesized that the degree of responses of the hydrolases might be due to biochemical heterogeneity among ICD. In this study the changes of intracellular lysosomal enzymes as well as Golgi enzymes including N-acetylglucosaminyl phosphotransferase (GlcNAcPTase) and extracellular hexosaminidase (HEX) were investigated using normal and ICD fibroblasts. Sucrose loading induced the activities of intracellular HEX and GlcNAcPTase simultaneously only in responding-type ICD cells, and not in nonresponding-type ICD cells, indicating that two biochemical heterogeneous groups exist in ICD.