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共同联系。最近，一个令人兴奋的主题出现了：如果突变蛋白的积累被消除，症状的进展不仅停止，而且还导致疾病的恢复。神经退行性疾病是可逆的第一个迹象来自多聚谷氨酰胺紊乱亨廷顿病（HD）的诱导型小鼠模型。在存在扩增的多聚谷氨酰胺亨廷顿蛋白的情况下，小鼠重现HD样症状。当突变基因表达被消除时，不仅聚集体消失，症状也消退了。这些发现表明，神经退行性疾病不再需要被视为死刑。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，用化学化合物库筛选设计良好的基于细胞的测定将使我们不仅能够进一步阐明哪种降解途径是重要的，而且还可能揭示这些途径可以被激活的新方法。因此，我们设计了一个功能性的细胞为基础的测定，不仅监测聚集的突变亨廷顿蛋白，但也清除。为此，我们创建了一种稳定的细胞系，其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，我们将带来神经退行性过程的恢复，如在该疾病的几个动物模型中所示。