Development of therapeutic measures for triplet repeat diseases

齿状核红核-苍白球路易氏体萎缩（DRPLA）是一种常染色体显性遗传的神经退行性疾病，其特征是小脑共济失调和伴随的神经系统症状的各种组合。在我们以前的研究中，我们发现DRPLA是由染色体12p13.31上DRPLA基因的CAG重复序列不稳定扩增引起的。目的探讨齿状核红核-苍白球路易氏体萎缩（DRPLA）的治疗方法，我们建立了一种培养系统，可以对DRPLA基因的多聚谷氨酰胺延伸段引起的毒性进行密度和定量分析。型多聚谷氨酰胺延伸导致核周以及核内聚集体形成。TUNEL法检测发现，具有聚集体的细胞经常发生凋亡。结果表明，处理突变DRPLA蛋白对于产生具有聚集体形成潜力的“有毒片段”非常重要。核包涵体的形成也被证实在小脑齿状核的神经元的尸检脑DRPLA患者，这证实了在DRPLA神经元变性的分子机制的核内聚集体的形成的作用。为了进一步研究聚集体形成的分子机制，我们测试了各种转氨酶抑制剂。我们发现，胱胺和单丹尸胺显着抑制聚集体的形成和凋亡细胞死亡。这些结果提出了转氨酶反应参与聚集体形成的可能性。总之，我们已经证明了一种新的策略，为DRPLA的治疗措施的发展。

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by various combinations of cerebellar ataxia and accompanying neurological symptoms. In our previous study, we discovered that DRPLA is caused by unstable expansion of a CAG repeat of the DRPLA gene on chromosome 12p13.31. To develop therapeutic measures for dentatorubral-pallidoluysian atrophy (DRPLA), we have developed a culture system which allow densitive and quantitative analysis of the toxicity caused by the expanded polyglutamine stretches of the gene for DRPLA.We discovered that expression of truncated DRPLA protein with expanded polyglutamine stretches but not of those with wild-type polyglutamine stretches results in formation of perinuclear as well as intranuclear aggregate formation. It was also found that the cells with the aggregate bodies frequently under go apoptotic cell death as measured by TUNEL assay. The results indicate that pocessing of mutant DRPLA protein is important to generate "toxic fragments" which have a potential for aggregate formation. Formation of nuclear inclusions was also confirmed in the neurons of cerebellar dentate nucleus of autopsied brains of patients with DRPLA, which confirms the role of intranuclear aggregate formation in the molecular mechanisms of neuronal degeneration in DRPLA.To further investigate the molecular mechanisms of aggregate formation, we tested various transglutaminase inhibitors. We found that cystamine and monodansylcadaverine significantly suppressed the aggregate formation and apoptotic cell death. The results raise the possibility that transglutaminase reaction is involved in the aggregate formation. Taken together, we have demonstrated a new strategy for the development of therapeutic measures for DRPLA.