The long-term goal of the proposed investigation is to study the pathobiology of genetic neurodegenerative disorders affecting infants and children and to explore the possible therapeutic means of such disorders. Using an authentic murine model of globoid cell leukodystrophy, twitcher mouse, we have been investigating the natural disease processes, with particular focus on cellular reactions associated with the progression of the pathology in the central as well as in the peripheral nervous systems. The studies proposed in this application are a continuation and/or extension of the ongoing studies in my laboratory. Twitcher is a murine model of a genetic demyelinating disease caused by the mutation of the enzyme galactosylceramidase. Although demyelination is a conspicuous histopathology clinical symptoms such as paralysis, opisthotonus and seizures noted in this mutant as well as in GLD patients are clearly attributable to neuronal dysfunction. We plan to investigate 1). the extent of neuronal pathology/dysfunction using the expression of the immediate early genes (IEGs) and/or heat shock proteins (HSPs) as markers and analysis of structural alterations with horseradish peroxidase (HRP) and/or with Golgi preparations. In twitcher, expression of major histocompatibility complex (MHC) class I and II molecules increases with the progression of demyelination. The twitcher mouse with class II-deficient background shows ameriolation of neuropathological lesions (66). 2). We hypothesize that the deficiency of MHC class I molecules also has some beneficial effects to lessen the twitcher pathology and so we will investigate a possible role of MHC class I molecules in twitcher pathology by generating the twitcher mouse with MHC class I deficient background (MHC class I knockout twitcher). And 3). we plan to explore the feasibility of gene transfer as treatment of genetic diseases using twitcher as a model. The studies include generation of transgenic twitcher mouse with the human galactosylceramidase cDNA under the transcriptional control of the myelin basic protein gene as well as the transfer of the human cDNA expression constructs into twitcher cells/tissues with in vitro, in vivo and ex vivo approaches. The result of these studies will be carefully monitored by morphology, immunocytochemistry and mRNA expression of myelin proteins assay of galactosylceramidase activity, analysis of the level of psychosine etc. For the treatment of genetic diseases, transfer of the missing gene into appropriate tissues/cells is an obvious treatment of choice. However, in practice, there are many problems in transferring genes, in particular for the treatment of genetic diseases affecting the nervous system. Therefore, well controlled studies using an established animal model of the disease is essential before any attempt of gene therapy in human patients.
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