Aging, Amyloidosis and levels of Transthyretin Oxidative Carbonylation

描述(申请人提供)：转甲状腺四聚体淀粉样变性是一种致命性蛋白质错误折叠疾病，其特征是从血浆高四聚体蛋白转甲状腺四聚体(TTR)衍生的聚集体和淀粉样纤维沉积在细胞外。衰老是这些疾病的老年型(野生型)和遗传型(突变型)的主要危险因素。它不是 了解衰老过程的哪些方面导致TTR淀粉样变性的发生。蛋白质羰化是一种常见的老年氧化修饰，它与阿尔茨海默氏症和帕金森氏症等与年龄相关的神经退行性疾病有关。我们先前在体外已经证实，重组羰化TTR(CAR-TTR)比未氧化的TTR更不稳定，有更高的聚集倾向。我们还证明，目前唯一正在进行临床试验的基于TTR四聚体稳定的治疗方法，对CAR-TTR的疗效明显低于非氧化性TTR。这些研究的含义是，如果CAR-TTR水平随着年龄的增加而增加，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。尽管已在健康人的血浆中检测到CAR-TTR，但尚无系统研究其丰度随年龄的变化或TTR淀粉样变性的存在。我们的工作假设是：1)血浆中CAR-TTR含量随年龄增加而增加；2)TTR淀粉样变性患者血浆中CAR-TTR含量高于健康对照组。我们的目标是确定血浆中CAR-TTR值的增加是否与年龄和/或明显的TTR型淀粉样变性有关。为了实现这些目标，我们将利用我们已经建立的方法来量化血浆中的总TTR值，并优化我们的工作方案，以具体量化相同样本中的CAR-TTR值。实验设计包括以下组：i)20岁至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组；iii)健康的年轻和老年过表达人野生型TtR的转基因小鼠；iv)过表达人类野生型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，以及已经证实的CAR-TTR比非氧化TTR更容易聚集的事实，将表明这种与年龄相关的氧化修饰可能在TTR聚集障碍的发生中发挥作用。它还将开辟一种新的治疗方法来预防疾病的发生，包括抗氧化剂治疗，以防止CAR-TTR的形成。我们将在即将发布的R01提案中正式测试因果假说，即CAR-TTR的形成与TTR淀粉样变性的发病有关，并在体内测试新的治疗策略。最后，我们的研究结果可能对理解其他与年龄相关的蛋白质聚集疾病的启动具有直接意义，在这些疾病中，前体淀粉样蛋白可能对相同类型的氧化修饰敏感。

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.