Aging, Amyloidosis and levels of Transthyretin Oxidative Carbonylation

描述（由申请方提供）：甲状腺素运载蛋白淀粉样变性是一种致死性蛋白质错误折叠疾病，其特征为来源于血浆同型四聚体蛋白质甲状腺素运载蛋白（TTR）的聚集体和淀粉样原纤维的细胞外沉积。衰老是这些疾病的老年（野生型）和遗传（突变TTR）形式的主要风险因素。不 已知老化过程的哪些方面导致TTR淀粉样变性的发作。 蛋白质羰基化是老年常见的氧化修饰，它与老年相关的神经退行性疾病如阿尔茨海默病和帕金森病有关。我们先前在体外确定重组羰基化TTR（car-TTR）比非氧化TTR更不稳定并且具有更高的聚集倾向。我们还证明，目前在临床试验中基于TTR四聚体稳定化的唯一疗法对汽车TTR的效率明显低于非氧化TTR。这些研究的意义是，如果car-TTR水平随着年龄的增长而增加，它们将促进淀粉样变性的发生;此外，四聚体稳定疗法可能不足以治疗这些疾病。虽然在健康个体的血浆中检测到了car-TTR，但关于其随年龄或TTR淀粉样变性的存在而变化的丰度，还没有系统的研究。我们的工作假设是：1）血浆中的Car-TTR含量随年龄增加而增加; 2）TTR淀粉样蛋白患者血浆中的Car-TTR含量高于健康对照组。我们的目标是确定血浆中car-TTR的增加是否与年龄和/或明显的TTR淀粉样变性相关。为了实现这些目标，我们将利用我们已经建立的方法来定量血浆中的总TTR，并优化我们的工作方案，以特异性定量相同样品中的car-TTR。实验设计包括以下组：i）20至72岁的健康人; ii）TTR淀粉样变性患者和年龄匹配的健康对照; iii）过表达人野生型TTR的健康年轻和年老转基因小鼠; iv）过表达人野生型TTR的转基因小鼠，有和没有组织学证实的TTR沉积。 发现car-TTR含量与年龄和/或TTR淀粉样变性的阳性诊断之间的相关性，以及已经确定的事实，即car-TTR比非氧化TTR更容易聚集，将表明这种类型的年龄相关的氧化修饰可能在TTR聚集障碍的发病中起作用。它还将开辟一种新的治疗方法，以防止疾病发作，包括抗氧化治疗，以防止汽车TTR的形成。我们将正式测试因果假说，即汽车TTR的形成是参与发病的TTR淀粉样变性，和新的治疗策略在体内即将到来的R 01提案。最后，我们的研究结果可以有直接的影响，了解其他年龄相关的蛋白质聚集性疾病的发生，其中前体淀粉样蛋白可能容易受到相同类型的氧化修饰。

DESCRIPTION (provided by applicant): The transthyretin amyloidoses are fatal protein misfolding disorders characterized by the extracellular deposition of aggregates and amyloid fibrils derived from the plasma homotetrameric protein transthyretin (TTR). Aging is a major risk factor for both the senile (wild-type) and genetic (mutant TTR) forms of these disorders. It is not known which aspects of the aging process contribute to the onset of the TTR amyloidoses. Protein carbonylation is an oxidative modification common of old age and it is associated with age-related neurodegenerative illnesses such as Alzheimer's and Parkinson's diseases. We previously established in vitro that recombinant carbonylated TTR (car-TTR) is less stable and has higher propensity to aggregate than non-oxidized TTR. We also demonstrated that the only therapy currently under clinical trials based on TTR tetramer stabilization is significantly less efficient for car-TTR than for non-oxidized TTR. The implications of these studies are that if car-TTR levels increase with age, they will promote the onset of the amyloidoses; moreover, tetramer stabilization therapy may not be sufficient to treat these disorders. Although car-TTR has been detected in plasma of healthy individuals, no systematic studies exist with respect to its changing abundance with age or with the presence of TTR amyloidoses. Our working hypotheses are: 1) Car-TTR content in plasma increases with age; 2) Car-TTR content in plasma is higher in TTR amyloid patients than in healthy controls. Our goals are to establish whether an increase in car-TTR in plasma is associated with age and/or with overt TTR amyloidoses. To accomplish these goals we will exploit our already established methods to quantify total TTR in plasma and optimize our working protocols to specifically quantify car-TTR in the same samples. The experimental design includes the following groups: i) Healthy humans aged 20 to 72 years; ii) Patients with TTR amyloidosis and age-matched healthy controls; iii) Healthy young and old transgenic mice over-expressing human wild-type TTR; iv) Transgenic mice over-expressing human wild-type TTR, with and without histologically confirmed TTR deposition. Finding a correlation between car-TTR content with age and/or the positive diagnosis of TTR amyloidosis, together with the already established fact that car-TTR is more prone to aggregation than non-oxidized TTR, will indicate that this type of age-related oxidative modification may play a role in the onset of the TTR aggregation disorders. It will also open a new therapeutic approach to prevent disease onset consisting in anti-oxidant therapy to preclude car-TTR formation. We will formally test the causal hypothesis, i.e. car-TTR formation is involved in the onset of the TTR amyloidoses, and the novel therapeutic strategy in vivo in a forthcoming R01 proposal. Finally, the results of our studies can have direct implications to the understanding of the initiation of other age-related diseases of protein aggregation where the precursor amyloidogenic proteins might be susceptible to the same type of oxidative modifications.