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

用于检测阿尔茨海默病和相关疾病中错误折叠蛋白寡聚体的荧光探针

基本信息

批准号：
10604908
负责人：
David E Kang
金额：
$ 20.04万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10604908
关键词：
APP-PS1 Address Alzheimer&apos s Disease Alzheimer&apos s disease brain Alzheimer&apos s disease model Alzheimer&apos s disease related dementia Amyloid Amyloid beta-Protein Animal Model Antibodies Binding Biological Biological Assay Biological Markers Categories Characteristics Chemicals Clinical Data Dependence Deposition Detection Development Disease Dyes Evolution Exhibits Fluorescence Fluorescent Dyes Fluorescent Probes Future Gentian Violet Goals Growth Human Image In Vitro Kinetics Label Laboratories Link Monitor Muramidase Mus Normal tissue morphology Oranges Outcome Pathogenicity Pathologic Pathology Patients Phase Population Positron-Emission Tomography Proliferating Protocols documentation Public Health Role Sampling Series Solid Solvents Specificity Stains Symptoms Techniques Testing Thioflavin S Tissues Transgenic Mice Transgenic Organisms Validation abeta oligomer amyloid formation animal tissue brain tissue cohort detection assay detection method experimental study imaging probe improved in vivo in vivo imaging in vivo monitoring indexing innovation insight mild cognitive impairment misfolded protein monomer novel protein oligomer response scaffold screening success tau Proteins tau aggregation

项目摘要

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.

用于检测阿尔茨海默病中错误折叠蛋白质寡聚物的荧光探针疾病及相关病症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出现之前的小寡聚物的形成和增殖密切相关明显的晚期原纤维和斑块。因此，β淀粉样蛋白寡聚体（AβOs）是最直接的用于监测 AD 发病和进展的生物标志物。然而，利用这种生物标志物的尝试已经由于缺乏可靠检测生物样品中 AβO 的技术而受到严重阻碍组织。虽然寡聚物选择性抗体为 AβO 提供了重要的见解，但它们的用途并没有扩展检测体内低聚物群体，更不用说监测它们的时间演变了。总体目标因此，该提案的目的是鉴定用于检测 AβO 的小寡聚物选择性染料并验证它们对 AD 动物模型组织和患者组织中的 AβO 和潜在相关寡聚体具有特异性。多个实验室观察到体内 Aβ 组装不仅表现出纯 S 形，而且还表现出双相 ThT 动力学。我们已经证明双相 ThT 动力学的发生与 ThT 的发生直接相关随着单体浓度的增加，前原纤维寡聚体数量迅速增加。在这里我们建议利用这种从基本上无寡聚物的 S 形到寡聚物主导的双相动力学的转变筛选一系列现成的荧光染料，以确定它们对 AβO 的选择性优于 AβF 和单体。这种方法的初步测试已经产生了一种非常有前途的候选染料。我们的初步数据还表明该染料可以特异性地对 AD 动物模型中的低聚物沉积物进行染色。虽然非常令人鼓舞，但我们当前的低聚物选择性染料的实用性需要进一步验证。在此外，我们寻求鉴定多种化学和结构不同的低聚物选择性染料，以改善有机会将其中之一开发成用于低聚物体内成像的 PET 探针。因此，我们将延长我们的目前从 AβO 选择性染料筛选到从不同染料类别中选择的更大组荧光染料。在与此同时，我们将仔细检查当前的染料是否能够可靠地检测疾病各个阶段的 AβO，以及在动物模型和患者组织中都是如此。通过我们的鉴定，有前途的新型 AβO 选择性染料筛选测定将对其在组织中的特异性进行相同的离体验证。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，用于开发离体检测 AβO 的新方法，以及，最重要的是，作为未来体内死前低聚物选择性 PET 探针的检测部分患者的寡聚物成像。