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

(1)伴伴治疗Pelizaeus-Merzbacher。我们未能建立一种候选化学物质的气相色谱测定方法。我们转而寻找降低蛋白脂蛋白（PLP）表达水平的化学物质。PLP重复占PMD患者的一半。在表达PLP的C6胶质瘤细胞系中，我们可以通过定量RT-PCR检测PLP mRNA水平。次黄嘌呤鸟嘌呤磷酸核糖基转移酶使PLP的表达正常化。首先，我们发现细胞分化越多，PLP表达越多。筛选条件优化如下：从食品中提取的化学物质，最终浓度为10μM，细胞密度为2.5×10^5/孔（9.2cm^2），添加DMEM（低糖）（含1%胎牛血清）48小时。我们筛选了包含一百多种食品化学物质的资料库。我们发现2种化学物质使PLP mRNA表达降低50%以上。有11种化学物质对PLP表达量的影响大于30%。我们已经发现，PLP缺失突变会导致细胞内蛋白生成系统过载，从而导致细胞死亡。因此，降低PLP水平的食品化学物质可能适用于PLP基因重复或缺失突变的PMD患者。(2) PMD小鼠模型疗效的建立。为了正确评价PMD药物，我们需要评估小鼠模型的髓鞘形成程度。我们试图通过显微镜检查定量测量髓鞘形成。首先，我们在常规HE、Kluver-Barrera染色或PLP抗体和髓鞘碱性蛋白免疫染色后，用光镜检查评估髓鞘形成程度。这些方法部分揭示了PMD模型小鼠的髓鞘发育异常。电镜检查显示髓鞘形成程度正常。在中枢神经系统中，视神经因其含有数量均匀的神经元和少突胶质细胞而具有较好的定量检测优势。少

(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