Fluorescent probes for detection of misfolded protein oligomers in Alzheimer's Disease and related disorders

检测阿尔茨海默病患者错折叠蛋白低聚物的荧光探针 疾病及相关障碍 有重要证据表明，阿尔茨海默病(AD)和相关疾病的临床症状 与小分子低聚物的形成和扩散密切相关，这些低聚物出现在 明显的晚期纤维和斑块。因此，淀粉样β寡聚体(AβOs)是最直接的 用于监测AD发病和进展的生物标志物。然而，利用这种生物标记物的尝试已经 由于缺乏可靠检测生物样品中AβOs的技术，以及 纸巾。虽然寡聚体选择性抗体为AβOs提供了重要的洞察力，但它们的用途并不广泛 检测体内的寡聚体种群，更不用说监测它们的时间进化了。总体目标 因此，这项建议的目的是鉴定用于检测AβO的小分子选择性染料，并验证 在AD动物模型组织和患者组织中，它们对AβOs和潜在相关寡聚体的特异性。 多个实验室观察到，在体内，β组装不仅表现出纯粹的乙型，而且还表现出 两相THT动力学。我们已经证明，双相tht动力学的开始与开始直接相关。 随着单体浓度的增加，前纤维齐聚物数量迅速增加。在这里我们 建议使用这种从基本上不含低聚物的S型转变为低聚物主导的两相动力学 筛选一系列容易获得的荧光染料，以考察它们对AβOs和AβF及其单体的选择性。 对这种方法的初步测试已经产生了一种非常有希望的染料候选者。我们的初步数据也 表明该染料能特异性地染色AD动物模型中的低聚物沉积。 虽然非常令人鼓舞，但我们目前的低聚物选择性染料的实用性还需要进一步验证。在……里面 此外，我们寻求鉴定多种化学和结构上不同的低聚物选择性染料，以改善 有机会将其中一种发展成PET探针，用于低聚物的体内成像。因此，我们将延长我们的 目前的筛网用于βO-选择性染料，以从不同的染料类别中选择更大的一组荧光染料。在……里面 同时，我们将仔细检查当前的染料是否在疾病的不同阶段可靠地检测到AβO，以及 在动物模型和患者组织中也是如此。一种前景看好的新型βO-选择性染料 筛选试验将在组织中接受同样的体外特异性验证。 我们预计，这些实验将产生多种有前景的AβO选择性染料，并应用于 低聚物形成的基础研究，用于开发体外检测AβOS的新方法，以及， 最重要的是，作为未来用于活体生前的低聚物选择性PET探针的检测部分 低聚物在患者体内的成像。

Fluorescent Probes for Detection of Misfolded Protein Oligomers in Alzheimer's Disease and Related Disorder There is significant evidence that the clinical symptoms of Alzheimer’s Disease (AD) and related disorders are closely linked to the formation and proliferation of small oligomers that precede the emergence of the prominent late-stage fibrils and plaques. Therefore, amyloid beta oligomers (AβOs) are the most direct biomarkers for monitoring the onset and progression of AD. Attempts at utilizing this biomarker, however, have been severely hampered by the dearth of techniques for the reliable detection of AβOs in biological samples and tissues. While oligomer-selective antibodies have provided important insights into AβOs, their use doesn't extend to detecting oligomer populations in vivo, let alone monitoring their temporal evolution. The overall objectives of this proposal are therefore to identify small oligomer-selective dyes for the detection of AβOs and to validate their specificity for AβOs, and potentially related oligomers, in tissues of animal models of AD and patient tissue. Multiple laboratories have observed that in vivo Aβ assembly displays not only purely sigmoidal but also biphasic ThT kinetics. We have shown that the onset of biphasic ThT kinetics directly correlates with the onset and rapid increase in prefibrillar oligomer populations with increases in monomer concentrations. Here we propose to use this transition from essentially oligomer-free sigmoidal to oligomer-dominated biphasic kinetics to screen a selection of readily available fluorescent dyes for their selectivity for AβOs over AβFs and monomers. An initial test of this approach already yielded a highly promising dye candidate. Our preliminary data also indicate that this dye specifically stains oligomer deposits in animal models of AD. While very encouraging, the utility of our current oligomer-selective dye requires further validation. In addition, we seek to identify multiple chemically and structurally distinct oligomer-selective dyes to improve the chances to develop one of them into a PET probe for in vivo imaging of oligomers. We will therefore extend our current screen for AβO-selective dyes to a larger set of fluorescent dyes selected from different dye categories. In parallel, we will scrutinize whether the current dye reliably detects AβOs at various stages of the disease, and does so in animal models as well as patient tissues. Promising novel AβO-selective dyes identified through our screening assay will be subjected to the same ex vivo validation of their specificity in tissues. We anticipate that these experiments will yield multiple promising AβO-selective dyes with application for fundamental studies of oligomer formation, for the development of new assays for detecting AβOs ex vivo, and, most importantly, as the detection moiety for a future oligomer-selective PET probe for antemortem in vivo oligomer imaging in patients.