Therapy for neurodegeneerative disease using novel screening method for chemical chaperon.

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(1)Therapy for Pelizaeus-Merzbacher with chaperon. We failed to establish the gas-chromatographically measurement for a candidate chemical. We switched to search for chemicals to decrease the expression level of proteolipid protein(PLP). Duplication of PLP accounts for the half of the PMD patients. In C6 glioma cell line expressing PLP, we could measure PLP mRNA level by Quantitative RT-PCR. PLP expression was normalized by that of hypoxanthine guanine phosphoribosyl transferase. At first, we found that the more cell differentiate, the more PLP expresses. We optimized screening condition as follows ; chemicals extracted from food were added at final concentration of 10μM at cell density of 2.5×10^5/well(9.2cm^2) for 48hrs with DMEM(low glucose) including 1%fetal bovine serum. We screened the library including more than one hundred of food chemicals. We found 2 chemicals that decrease the PLP mRNA expression more than 50%. 11 chemicals were found to decrease the PLP expression level mor … More e than 30%. We have already found that PLP missence mutations cause overload to proteorytic system in the cell leading to cell death. Therefore, food chemicals that lower the level of PLP may be applied to the PMD patients with duplication as well as missence mutations in PLP gene.(2)Establishment of therapeutic effect in mouse model of PMD. For proper evaluation of drugs for PMD, we need to assess the degree of myelination in mouse model. We tried to quantitatively measure myelination by microscopic examinations. Firstly, we evaluated the degree of myelination using light microscopic examinations after conventional HE, Kluver-Barrera's or immune staining using the antibodies to PLP and myelin basic protein. These methods partially disclosed the dysmyelination of PMD model mouse. Electron microscopic examination revealed the degree of myelination properly. Optical nerve was superior for quantitative examination among central nervous system, as it contained the uniform numbers of neuron and oligodendrocytes. Less