Aggregation and Clearance of Mutant Huntingtin(RMI)

突变亨廷顿蛋白（RMI）的聚集和清除

• 批准号: 7057708
• 负责人: JAMES ROTHMAN
• 金额: $ 0.48万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-16 至 2006-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7057708
• 关键词: Huntington' s disease; cell line; chemical aggregate; confocal scanning microscopy; gene mutation; green fluorescent proteins; high throughput technology; homopeptide; nerve /myelin protein; peptide library; protein degradation; protein protein interaction

DESCRIPTION (provided by applicant): Accumulation and aggregation of mutant proteins are a common link across a wide array of neurodegenerative disorders. Recently, an exciting theme has emerged: if mutant protein accumulation is eliminated, symptomatic progression not only halts but also leads to recovery from disease. The first indication that neurodegenerative diseases are reversible comes from an inducible mouse model of the polyglutamine disorder Huntington's disease (HD). In the presence of expanded polyglutamine huntingtin, mice recapitulated HD-like symptoms. When mutant gene expression was abolished, not only did the aggregates disappear, the symptoms regressed. These findings signal that neurodegenerative diseases need no longer be considered a death sentence. Unfortunately, however, the pathway underlying clearance of these mutant proteins are not yet clear. We believe that screening a well-designed cell-based assay with a chemical compound library will allow us to not only further clarify which degradative pathway is important, but may also reveal new means by which these pathways can be activated. We have therefore designed a functional cell-based assay that monitors not only aggregation of mutant huntingtin protein, but also its clearance. To do so, we created a stable cell line that conditionally expresses the N-terminus of huntingtin protein with polyQ proteins of different polyQ lengths fused to variants of GFP. These cell lines permit high throughput confocal microscopy to examine the state of the expressed mutant protein in live cells. We hypothesize that by eliminating the accumulated protein we will bring about recovery of the neurodegenerative process, as shown in several animal models of the disease.

描述（由申请人提供）：突变蛋白的积累和聚集是多种神经退行性疾病的常见联系。最近出现了一个令人兴奋的主题：如果突变蛋白的积累被消除，症状的进展不仅会停止，而且还会导致疾病康复。神经退行性疾病是可逆的第一个迹象来自多聚谷氨酰胺疾病亨廷顿氏病 (HD) 的诱导小鼠模型。在存在扩展的聚谷氨酰胺亨廷顿蛋白的情况下，小鼠重现了HD样症状。当突变基因的表达被废除时，不仅聚集物消失了，症状也消退了。这些发现表明神经退行性疾病不再被视为死刑。然而不幸的是，这些突变蛋白的清除途径尚不清楚。我们相信，利用化合物库筛选精心设计的基于细胞的检测方法不仅可以使我们进一步阐明哪种降解途径是重要的，而且还可能揭示激活这些途径的新方法。因此，我们设计了一种基于细胞的功能测定，不仅可以监测突变亨廷顿蛋白的聚集，还可以监测其清除情况。为此，我们创建了一个稳定的细胞系，该细胞系条件性地表达亨廷顿蛋白的 N 末端，其中不同 PolyQ 长度的 PolyQ 蛋白与 GFP 变体融合。这些细胞系允许高通量共聚焦显微镜检查活细胞中表达的突变蛋白的状态。我们假设，通过消除积累的蛋白质，我们将实现神经退行性过程的恢复，如该疾病的几种动物模型所示。