Mechanisms of Toxicity in C. elegans Models of Transthyretin Amyloidosis

 描述（由申请方提供）：蛋白质聚集是大多数神经退行性疾病的特征，包括甲状腺素运载蛋白（TTR）淀粉样变性。TTR是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淀粉样变性中的外周神经变性是由限速TTR四聚体解离、异常单体错误折叠和错误折叠的TTR组装成一系列TTR聚集体结构引起的。细胞外聚集导致组织中的蛋白毒性，该组织不通过细胞非自主过程合成TTR，我们试图通过所提出的实验来理解该过程，并且对于任何聚集相关的神经退行性疾病都不理解该过程。在人类中，WT TTR聚集导致心肌病，而其他TTR突变的聚集导致原发性神经病。在此，我们报告的TTR淀粉样变性的转基因秀丽隐杆线虫模型的开发和部分表征，表现出TTR聚集和三个细胞的非自主可定量的神经元TTR蛋白毒性相关的细胞和亚细胞表型与人类疾病直接相关。我们将描述TTR mRNA水平和TTR构象，包括四聚体，和错误折叠的TTR寡聚体在这些模型中作为老化的函数，并将这些与观察到的神经元表型。基于微管的运输缺陷似乎是TTR蛋白毒性的重要机制特征。新的小分子和遗传工具的可用性来量化TTR构象，以及在同一活蠕虫中将单个神经元中的亚细胞表型成像为衰老的函数以量化表型变化的能力，为我们提供了了解神经变性的细胞生物学和生物化学的非凡机会。获得药物tafamaltine，它大大减缓了人类TTR淀粉样变性的进展，也将使我们能够辨别TTR聚集的抑制如何改变这些模型中明显潜在的神经变性的细胞生物学和生物化学缺陷。为了进一步了解TTR蛋白毒性在细胞和分子水平的机制，我们将寻找TTR蛋白毒性的调节剂，通过识别我们的TTR模型之一在无偏正向遗传筛选中表现出的运动缺陷的抑制因子。我们已经在中试筛选中鉴定了候选抑制因子，并显示一些抑制因子表现出适当的TTR合成和分泌，这表明该筛选可以鉴定组织特异性分子靶标，这些分子靶标是细胞外TTR聚集体形成和细胞毒性之间的中继。这些研究将建立TTR C。线虫模型与细胞非自主TTR毒性研究相关。

 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.