Elucidation of molecular mechanisms of spinocerebellar degeneration through positional cloning of causative genes and development of the animal models.

为了阐明遗传性脊髓小脑共济失调中神经元变性的分子机制，并制定治疗这些疾病的措施，我们试图建立齿状小脑-苍白球样萎缩（DRPLA）的动物模型，这是遗传性脊髓小脑变性的一种形式。作为第一步，我们已经分离并确定了DRPLA全长人和小鼠cDNA克隆的详细结构。此外，我们还分离了人类和小鼠的DRPLA基因组DNA。我们发现DRPLA基因不仅在中枢神经系统中广泛表达，而且在躯体器官中也广泛表达。我们发现，在DRPLA患者的中枢神经系统中，扩增的CAG重复序列存在相当大的体细胞嵌合体，这表明扩增的CAG重复序列存在体细胞不稳定性。为了创建动物模型，我们采取了两种策略。作为一种策略，我们从DRPLA患者克隆了一个具有最长（78个重复）CAG重复序列的基因组克隆，并将其插入具有可选择标记的胚胎干细胞（ES细胞）中。选择含有单拷贝DRPLA突变基因的胚胎干细胞，获得嵌合小鼠。现在正在尝试种系传播。作为另一种策略，我们创建了携带扩增CAG重复序列的突变体人DRPLA cDNA。制备了携带多份突变体DRPLA cDNA的转基因小鼠，并对其表型进行了研究。这两种动物模型的建立对进一步研究DRPLA的分子机制和制定DRPLA的治疗措施具有重要意义。

To elucidate molecular mechanisms of neuronal degeneration in hereditary spinocerebellar ataxia, and to develop therapeutic measures for these diseases, we have attempted to create animal models for dentatorubral-pallidoluysian atrophy (DRPLA), a form of hereditary spinocerebellar degenerations . As the first step, we have isolated and determined the detailed structures of full length human and mouse cDNA clones for DRPLA .Furthermore, we have also isolated human and mouse genomic DNA for DRPLA . We have revealed that the DRPLA gene is widely expressed not only in the central nervous system but also in the somatic organs . We have found that there is a considerable somatic mosaicism of the expanded CAG repeats in the central nervous system of DRPLA patients, suggesting the somatic instability of the expanded CAG repeats . To create animal models, we have undertaken two strategies . As one strategy, we have cloned a genomic clone from a DRPLA patient with the longest (78 repeat) CAG repeat, which was inserted into embryonic stem cells (ES cells) with a selectable marker . ES cells containing single copy of mutant DRPLA gene were selected and chimeric mice was obtained . Germline transmission is being attempted now . As another strategy, we have created mutant human DRPLA cDNA carrying the expanded CAG repeat . Transgenic mouse carrying multiple copies of the mutant DRPLA cDNA were made, and the phenotype is being investigated . These two lines of animal models should be highly useful for the future study of the molecular mechanisms of DRPLA,and to develop therapeutic measure for DRPLA .