Protein Trafficking in Neurodegenerative Diseases

描述(申请人提供)：突变蛋白的积累和聚集是不同神经退行性疾病的共同特征，例如多谷氨酰胺扩张性疾病亨廷顿病(HD)。最近出现的一个主题是，如果突变蛋白的积累被消除，症状进展不仅会停止，而且还会恢复。例如，在一个可诱导的亨廷顿病模型中，有症状的动物中突变蛋白积累的丢失导致了类似疾病的症状完全逆转。因此，如果我们能够加速清除一种致病突变蛋白，就存在着从疾病中恢复的诱人可能性。 但是细胞如何清除这些突变的蛋白质呢？突变蛋白的清除如何导致症状的恢复？为了深入了解这些问题，我们在携带突变亨廷顿蛋白的稳定转基因细胞系上运行了Affymetrix基因阵列。不同基因图谱的比较显示，表明溶酶体介导的降解和囊泡运输的途径发生了令人惊讶的强劲变化。这两个领域在亨廷顿氏病和多谷氨酰胺病中通常很少被探索，因此是一个丰富的问题来源。因此，在这个方案中，我们将系统地检验以下假设：1)溶酶体介导的降解对突变的亨廷顿蛋白的降解有显著影响；2)聚集导致囊泡运输的可逆缺陷。结合使用生化和遗传技术，我们还建议使用基于细胞的功能分析来识别蛋白质聚集和清除的调节因子：一种稳定的细胞系，它有条件地表达融合到绿色荧光蛋白变体的突变蛋白。总之，在这一资助期间，我们将揭示直接改变细胞中突变蛋白质水平的靶点，阐明处理这些困难蛋白质的关键基本降解途径，并研究蛋白质降解缺陷如何改变细胞中的囊泡运输。

DESCRIPTION (provided by applicant): Accumulation and aggregation of mutant proteins are common traits across different neurodegenerative disorders, such as the polyglutamine expansion disorder Huntington's disease (HD). A recently emerging theme is that if mutant protein accumulation is eliminated, symptomatic progression not only halts but also recovers. For example, in an inducible model of Huntington's disease, loss of mutant protein accumulation in symptomatic animals led to complete reversion of the disease-like symptoms. Therefore, if we can accelerate the clearance of a disease-causing mutant protein, there exists the tantalizing possibility of recovery from disease. But how do cells clear these mutant proteins? And how does clearance of the mutant proteins lead to recovery from symptoms? To gain insight into these questions we have run Affymetrix gene arrays on stably transfected cell lines that carry mutant huntingtin protein. The comparison of the different genetic profiles revealed surprisingly robust changes in pathways indicating lysosome-mediated degradation and vesicular trafficking. These two areas are little explored in Huntington's disease and polyglutamine diseases in general, and is thus a rich source of questions. In this proposal we will therefore systematically test the following hypotheses: 1) Lysosome-mediated degradation has a significant impact on the degradation of mutant huntingtin proteins; and 2) Aggregation leads to reversible deficits in vesicular trafficking. Using a combination of biochemical and genetic techniques we also propose to identify regulators of protein aggregation and clearance using a functional cell-based assay: a stable cell line that conditionally expresses mutant proteins fused to variants ol GFP. In sum, during this grant period we will reveal targets that directly alter the level of mutant proteins in a cell, elucidate the basic degradation pathways crucial for handling these difficult proteins, and examine how deficits in protein degradation alters vesicular trafficking in the cell.