Development of therapeutic measures for triplet repeat diseases

齿状体-苍白球萎缩症（DRPLA）是一种常染色体显性神经退行性疾病，以小脑共济失调和伴随的神经系统症状的各种组合为特征。在我们之前的研究中，我们发现DRPLA是由染色体12p13.31上DRPLA基因的CAG重复扩增不稳定引起的。为了开发齿状网膜-苍白球萎缩症（DRPLA）的治疗措施，我们开发了一种培养系统，可以对DRPLA基因扩展的聚谷氨酰胺延伸引起的毒性进行密集和定量分析。我们发现，扩增的聚谷氨酰胺延伸的截断DRPLA蛋白的表达，而不是野生型聚谷氨酰胺延伸的表达，导致核周围和核内聚集体的形成。TUNEL实验还发现，具有聚集体的细胞经常发生凋亡细胞死亡。结果表明，突变体DRPLA蛋白的加工对于产生具有聚集形成潜力的“有毒片段”是重要的。在DRPLA患者脑解剖后的小脑齿状核神经元中也发现了核包涵体的形成，这证实了核内聚集体的形成在DRPLA神经元变性的分子机制中的作用。为了进一步研究聚集体形成的分子机制，我们测试了各种转谷氨酰胺酶抑制剂。我们发现半胱胺和单胺尸胺能显著抑制聚集体的形成和凋亡细胞的死亡。这些结果提出了转谷氨酰胺酶反应参与聚集体形成的可能性。综上所述，我们已经展示了一种开发DRPLA治疗措施的新策略。

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.