Mechanisms of Toxicity in C. elegans Models of Transthyretin Amyloidosis

 描述(申请人提供)：蛋白质聚集是大多数神经退行性疾病的特征，包括转甲状腺蛋白(TTR)淀粉样变性。Ttr是一种由肝脏分泌到血液中的四聚体蛋白。令人信服的证据表明，TTR淀粉样变性的外周神经变性是由于限速TTR四聚体解离、异常单体错误折叠和错误折叠TTR组装成一系列TTR聚集体结构造成的。细胞外聚集导致组织中的蛋白毒性，而不是通过一种细胞非自治过程来合成TTR，我们试图通过拟议的实验来了解这一过程，并且还不知道这是否会导致任何聚集相关的神经退行性疾病。在人类中，WT TTR聚集会导致心肌病，而其他TTR突变的聚集会导致原发神经病。在这里，我们报告了转基因秀丽线虫TTR淀粉样变性模型的建立和部分特征，该模型显示了TTR聚集和三种与人类疾病直接相关的细胞和亚细胞表型非自主可量化的神经元TTR蛋白毒性相关的细胞和亚细胞表型。我们将在这些模型中表征TTRmRNA水平和TTR四聚体和错误折叠的TTR寡聚体的构象，并将其与观察到的神经元表型联系起来。基于微管的运输缺陷似乎是TTR蛋白毒性的一个重要机制特征。新的小分子和基因工具的可获得性来量化TTR构象，以及能够成像单个神经元中的亚细胞表型作为同一活虫衰老的函数来量化表型变化的能力，为我们提供了一个了解神经退行性变的细胞生物学和生物化学的非凡机会。获得药物他法米迪，大大减缓人类TTR淀粉样变性的进展，也将使我们能够辨别抑制TTR聚集是如何改变这些模型中明显导致神经变性的细胞生物学和生化缺陷的。为了在细胞和分子水平上进一步了解TTR蛋白毒性的机制，我们将通过在无偏正向遗传筛选中识别我们的一个TTR模型所表现出的运动缺陷的抑制因子来寻找TTR蛋白毒性的调节器。我们已经在中试筛选中确定了候选抑制子，并表明其中一些显示了适当的TTR合成和分泌，这表明该筛选可以识别组织特异的分子靶点，这些分子靶标是细胞外TTR聚集体的形成和细胞毒性之间的中继者。这些研究将建立与细胞非自主TTR毒性研究相关的TTR线虫模型。

 DESCRIPTION (provided by applicant): Protein aggregation is a feature of most neurodegenerative diseases, including the transthyretin (TTR) amyloidoses. TTR is a tetrameric protein secreted into the blood by the liver. Compelling evidence suggests that peripheral neurodegeneration in the TTR amyloidoses results from rate- limiting TTR tetramer dissociation, aberrant monomer misfolding and misfolded TTR assembly into a spectrum of TTR aggregate structures. Extracellular aggregation leads to proteotoxicity in tissues not synthesizing TTR by a cell non-autonomous process that we seek to understand via the proposed experiments, and which is not understood for any aggregation-associated neurodegenerative disease. In humans, WT TTR aggregation leads to a cardiomyopathy, whereas aggregation of other TTR mutations leads to a primary neuropathy. Herein, we report the development and partial characterization of transgenic Caenorhabditis elegans models of the TTR amyloidoses, exhibiting TTR aggregation and three cell non-autonomous quantifiable neuronal TTR proteotoxicity-associated cellular and sub-cellular phenotypes with direct relevance to human disease. We will characterize TTR mRNA levels and TTR conformations including tetramers, and misfolded TTR oligomers in these models as a function of aging and correlate these with the neuronal phenotypes observed. Defects in microtubule-based trafficking appear to be a centrally important mechanistic feature of TTR proteotoxicity. The availability of novel small molecule and genetic tools to quantify TTR conformation, as well as the ability to image sub-cellular phenotypes in a single neuron as a function of aging in the same living worm to quantify phenotypic changes affords us an extraordinary opportunity to understand the cell biology and biochemistry of neurodegeneration. Access to the drug tafamidis, which dramatically slows progression of the TTR amyloidoses in humans will also allow us to discern how inhibition of TTR aggregation alters the cell biological and biochemical defects apparently underlying neurodegeneration in these models. To further understand the mechanisms of TTR proteotoxicity at the cellular and molecular level, we will search for modulators of TTR proteotoxicity by identifying suppressors of a locomotion defect exhibited by one of our TTR models in a unbiased forward genetic screen. We have identified candidate suppressors in a pilot screen and showed that some exhibited proper TTR synthesis and secretion, suggesting that this screen could identify tissue specific molecular targets that are relays between the formation of extracellular TTR aggregates and cellular toxicity. These studies will establish TTR C. elegans models as relevant to the study of cell non-autonomous TTR toxicity.