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.     

β-Glucosidase and β-Galactosidase in Primary Cultures of Rat Astrocytes: Comparison to the Brain Enzymes

In primary astrocyte cultures beta-glucosidase (EC 3.2.1.21) and beta-galactosidase (EC 3.2.1.23) showed pH optima and Km values identical to rat brain enzymes, using methylumbelliferyl glycosides and labeled gluco- and galactocerebrosides as substrates. The activities of both glycosidases increased in culture up to 3-4 weeks. In rat brain only galactosidase increased; glucosidase activity declined between 12-20 days after birth. The specific activities were two- to sixfold higher in astrocyte cultures than in rat brain. These activities were not due to uptake of enzymes from the growth medium. Secretion of beta-galactosidase, but not beta-glucosidase nor acid phosphatase could be demonstrated. These results support the suggestion of a degradative function for astrocytes in the brain.     

Transduction of Cultured Oligodendrocytes from Normal and Twitcher Mice by a Retroviral Vector Containing Human Galactocerebrosidase (GALC) cDNA

Krabbe disease or globoid cell leukodystropy is a lysosomal disorder caused by a deficiency of galactocerebrosidase (GALC) activity. This results in defects in myelin that lead to severe symptoms and early death in most human patients and animals with this disease. With the cloning of the GALC gene and the availability of the mouse model, called twitcher, it was important to evaluate the effects of providing GALC via a retroviral vector to oligodendrocytes in culture. After differentiation, the untransduced cells from normal mice extended highly branched processes while those from the twitcher mice did not. oligodendrocytes in culture can be readily transduced to produce much higher than normal levels of GALC activity. Transduced normal and twitcher cells formed clusters when plated at high density. Transduction of twitcher oligodendrocytes plated at lower density, followed by differentiation, resulted in some cells having a completely normal appearance with highly branched processes. Other cells showed retraction and fragmentation. Perhaps over expression of GALC activity may be detrimental to oligodendrocytes. These studies demonstrate that the phenotype of twitcher oligodendrocytes can be corrected by providing GALC via gene transfer, and this could lead the way to future studies to treat this disease.     

Formation of Free Fatty Acid and Ceramide During Brain Handling: Lability of Sphingomyelin

Intact brain and brain homogenates readily form free fatty acids and ceramides, even in the cold during subcellular isolation procedures. The fatty acid formation is slightly stimulated by chelators and might be due to phospholipid hydrolysis by lysosomal phospholipases. The ceramide formation is accompanied by loss of sphingomyelin and is apparently due to the action of neutral, metal ion-activated sphingomyelinase. The latter reaction is inhibited by EDTA whereas both degradative processes are inhibited by mercuriphenylsulfonate, the thiol-reacting inhibitor. Cerebroside does not seem to be a source of accumulated ceramide.