Protein Trafficking in Neurodegenerative Diseases

描述（由申请人提供）：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，例如多聚谷氨酰胺扩增障碍亨廷顿病（HD）。最近出现的一个主题是，如果突变蛋白质的积累被消除，症状的进展不仅停止，而且恢复。例如，在亨廷顿病的诱导模型中，有症状的动物中突变蛋白积累的丧失导致疾病样症状的完全逆转。因此，如果我们能够加速清除致病突变蛋白，就存在着从疾病中恢复的诱人可能性。 但是细胞是如何清除这些突变蛋白的呢？突变蛋白的清除是如何导致症状恢复的？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染的细胞系上运行了Affyrin基因阵列。不同遗传谱的比较揭示了指示溶酶体介导的降解和囊泡运输的途径中令人惊讶的稳健变化。这两个领域在一般的亨廷顿病和多聚谷氨酰胺疾病中很少被探索，因此是一个丰富的问题来源。因此，在本提案中，我们将系统地测试以下假设：1）溶酶体介导的降解对突变亨廷顿蛋白的降解具有显著影响;和2）聚集导致囊泡运输的可逆缺陷。使用生物化学和遗传技术的组合，我们还提出了确定调节蛋白质聚集和清除使用功能性细胞为基础的测定：一个稳定的细胞系，有条件地表达突变体蛋白融合的变体或绿色荧光蛋白。总之，在此资助期间，我们将揭示直接改变细胞中突变蛋白水平的靶点，阐明处理这些困难蛋白的基本降解途径，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.