Two major projects are proposed to continue for two more years my life- long research interest in the neurogenetic disorders.. He first project concerns a cross-breeding experiment between two mouse mutants of lysosomal beta-galactosidases, the twister mouse (galactosylceramidase deficiency, model of human Krabbe disease) and acid beta-galactosidase knockout mouse we recently generated (model of GM-gangliosidosis). This already ongoing experiment has yielded a totally unexpected and paradoxical results in addition to the anticipated massive accumulation of lactosylceramide. The observations are so contrary to the conventional wisdom concerning autosomal recessive disorders that they must be followed up in order to eventually clarify the underlying genetic and metabolic mechanism. The essence of the paradoxical findings is that twitcher mice with additional acid beta-galactosidase deficiency have by far the mildest phenotype, followed by twitcher mice with the normal complement of two acid beta-galactosidase genes and that the twitcher mice with a single functional aid beta-galactosidase gene have the most severe disease with additional neuronal lesions not seen in any other genotypes. It should also be noted that we compare only offspring of double-carrier mating in the same experiment in order to exclude variations in the genetic background of the mice. Detailed analytical studies related to galactosylceramide, ganglioside and related compounds, including psychosine (galactosyl-sphingosine) are proposed to lay a solid ground for future studies. The possibility that accumulation that accumulation of GM1- ganglioside in the doubly deficient mice somehow counteracts the apoptotic effect of psychosine will be tested in cultured embryonic mouse fibroblasts. The second major project is to generate mouse mutants with point mutations in the sphingolipid activator proteins A and D (sap, saposin A, D) respectively. Despite may studies in vitro and cell cultures, we still need the ultimate test for the essentiality of these activator proteins in the whole body. We have already generated the necessary targeting vectors which introduce point mutations in the sap A and D domains of the ES cell sap precursor gene with the use of the Cre- loxP system. The point mutations to be introduced are (1) to abolish the glycosylation site, and (2) to abolish one of the six cysteine residues. These point mutations are known in sap B and C in humans causing clinical diseases due to deficiency of the respective activator proteins without affecting processing of other domains.
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