ConditionalLoxP-flanked glucosylceramide synthase allele controlling glycosphingolipid synthesis

Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
genesis (Impact Factor: 2.02). 12/2005; 43(4):175-80. DOI: 10.1002/gene.20167
Source: PubMed


Glycosphingolipids are organizational building blocks of plasma membranes that participate in key cellular functions, such as signaling and cell-to-cell interactions. Glucosylceramide synthase--encoded by the Ugcg gene--controls the first committed step in the major pathway of glycosphingolipid synthesis. Global disruption of the Ugcg gene in mice is lethal during gastrulation. We have now established a Ugcg allele flanked by loxP sites (floxed). When cre recombinase was expressed in the nervous system under control of the nestin promoter, the floxed gene underwent recombination, resulting in a substantial reduction of Ugcg expression and of glycosphingolipid ganglio-series levels. The mice deficient in Ugcg expression in the nervous system show a striking loss of Purkinje cells and abnormal neurologic behavior. The floxed Ugcg allele will facilitate analysis of the function of glycosphingolipids in development, physiology, and in diseases such as diabetes and cancer.

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    • "Miglustat but not eliglustat tartrate is able to traverse the blood-brain barrier and thus might be used to treat the neuropathic glycosphingolipidoses. While excessive inhibition of the glycosphingolipid biosynthetic pathway could be detrimental to neuronal development and stabilization [10]–[12], the goal is to effect a partial reduction such that the rate of synthesis is matched by the residual capacity of the cells to degrade the substrates. Accordingly, SRT is arguably best suited for those indications that retain some measure of residual enzyme activity as in type 3 Gaucher patients and late-onset Tay-Sachs diseases. "
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    ABSTRACT: The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects.
    PLoS ONE 06/2011; 6(6):e21758. DOI:10.1371/journal.pone.0021758 · 3.23 Impact Factor
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    • "Knockout (KO) of the gene in mice results in embryonic death at E9.5, and findings strongly indicate that the enzyme plays key roles in development and morphogenesis of multicellular organisms (Yamashita et al. 1999). The nervous, tissue-specific knocking out of the enzyme in mice has also been assessed, showing that it is also essential in mouse brain development (Jennemann et al. 2005; Yamashita et al. 2005). Previous work also has found that in Drosophila melanogaster, the enzyme is essential and involved in apoptosis by controlling ceramide content (Kohyama-Koganeya et al. 2004). "
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    ABSTRACT: Ceramide glucosyltransferase (Ugcg) [uridine diphosphate (UDP)-glucose:N-acylsphingosine D-glucosyltransferase or UDP-glucose ceramide glucosyltransferase (GlcT): EC] catalyzes formation of glucosylceramide (GlcCer) from ceramide and UDP-glucose. There is only one Ugcg gene in the mouse genome, which is essential in embryogenesis and brain development. The nematode Caenorhabditis elegans has three Ugcg genes (cgt-1, cgt-2 and cgt-3), and double RNAi of the cgt-1 and cgt-3 genes results in lethality at the L1 larval stage. In this study, we isolated knockout worms for the three genes and characterized the gene functions. Each gene product showed active enzymatic activity when expressed in GM95 cells deficient in glycosphingolipids (GSLs). When each gene function was disrupted, the brood size of the animal markedly decreased, and abnormal oocytes and multinucleated embryos were formed. The CGT-3 protein had the highest Ugcg activity, and knockout of its gene resulted in the severest phenotype. When cgt-3 RNAi was performed on rrf-1 worms lacking somatic RNAi machinery but with intact germline RNAi machinery, a number of abnormal oocytes and multinucleated eggs were observed, although the somatic phenotype, i.e., L1 lethal effects of cgt-1/cgt-3 RNAi, was completely suppressed. Cell surface expression of GSLs and sphingomyelin, which are important components of membrane domains, was affected in the RNAi-treated embryos. In the embryos, an abnormality in cytokinesis was also observed. From these results, we concluded that the Ugcg gene is indispensable in the germline and that an ample supply of GlcCer is needed for oocytes and fertilized eggs to maintain normal membranes and to proceed through the normal cell cycle.
    Glycobiology 02/2011; 21(6):834-48. DOI:10.1093/glycob/cwr019 · 3.15 Impact Factor
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    • "bp were indicative of the wildtype , floxed, and null alleles, respectively (Yamashita et al., 2005a). The constitutive L7Cre transgene was detected using the following primers: L7Cre forward, 5 0 -AGGTTCGTTCACTCATGGA-3 0 ; and L7Cre reverse, 5 0 -TCGACCAGTTTAGTTACCC-3 0 . "
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    ABSTRACT: Glycosphingolipids (GSLs) occur in all mammalian plasma membranes. They are most abundant in neuronal cells and have essential roles in brain development. Glucosylceramide (GlcCer) synthase, which is encoded by the Ugcg gene, is the key enzyme driving the synthesis of most neuronal GSLs. Experiments using conditional Nestin-Cre Ugcg knockout mice have shown that GSL synthesis in vivo is essential, especially for brain maturation. However, the roles of GSL synthesis in mature neurons remain elusive, since Nestin-Cre is expressed in neural precursors as well as in postmitotic neurons. To address this problem, we generated Purkinje cell-specific Ugcg knockout mice using mice that express Cre under the control of the L7 promoter. In these mice, Purkinje cells survived for at least 10-18 weeks after Ugcg deletion. We observed apparent axonal degeneration characterized by the accumulation of axonal transport cargos and aberrant membrane structures. Dendrites, however, were not affected. In addition, loss of GSLs disrupted myelin sheaths, which were characterized by detached paranodal loops. Notably, we observed doubly myelinated axons enveloped by an additional concentric myelin sheath around the original sheath. Our data show that axonal GlcCer-based GSLs are essential for axonal homeostasis and correct myelin sheath formation.
    Glia 08/2010; 58(10):1197-207. DOI:10.1002/glia.20999 · 6.03 Impact Factor
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