NOVEL EXOSOME BIOMARKERS OF IRON PATHOLOGY IN AD

项目摘要/摘要 阿尔茨海默病(AD)困扰着数以百万计的美国人，但目前还没有有效的治疗方法。铁已经被证明是 参与AD的关键病理过程，包括淀粉样蛋白和tau聚集、炎症、氧化 压力和细胞死亡机制。尽管有越来越多的证据，但要确定 体内的铁代谢，限制了生物标记物和新疗法的潜在转化。外切体是 由细胞释放的纳米大小的囊泡，用于运输蛋白质、核酸、金属、脂类或代谢物。而当 Exosome反映细胞过程，并可以揭示人类组织和生物液中与疾病相关的病理， AD外切体中铁的异常还没有被研究。我们将通过以下方式解决这一知识差距 最先进的外切体分离技术结合先进的铁成像、蛋白质定量和 下一代测序方法。我们的目标是研究外周血铁的失调。 死亡的AD大脑，以揭示AD的特异性生物标志物，促进新疗法的开发。 该项目的目标是：(1)确定胞外体的数量、氧化状态和细胞来源 铁在公元后会发生变化。使用核磁共振和同步辐射X射线显微镜，我们将量化组织铁含量和 人AD和对照海马区标本中的氧化状态。然后我们将使用我们的小说exosome 隔离平台，外来，从同一海马铁含量高的区域分离外小体 标本。使用针对细胞表面蛋白的抗体，我们将根据以下条件丰富分离的外切体 它们的细胞起源(如神经元、小胶质细胞等)。我们将对每种细胞类型的胞外铁含量进行量化 采用电感耦合等离子体质谱(ICPMS)，并用电子显微镜检测胞外铁氧化状态。 总之，我们将确定阿尔茨海默氏症患者的铁含量和氧化状态是否发生了变化 与对照组相比，尤其是起源于小胶质细胞(大脑的免疫细胞)的外切体。 (2)检测AD外切体中铁相关蛋白和RNA的异常表达。使用Western blotting在 我们将确定在铁代谢中起作用的蛋白质水平是否发生了变化 AD组与对照组比较。因为外体通常富含已知的调节 基因表达，我们将使用RNA-Seq来确定外体microRNAs是否调控这些相同的 铁相关蛋白在AD中也会发生改变。机器学习算法将使创建一份地图集 连接铁、铁相关蛋白和神经病理学的microRNAs，这应该提供更深层次的 对AD生物学的理解。 阿尔茨海默病脑内外切体含量的特征应该导致铁的细胞特异性特征 与神经退行性变相关的调节失调。这种方法可能会阐明AD生物学的新方面， 导致新的检测方法来检测早期阿尔茨海默病，并促进急需的未来治疗。

PROJECT SUMMARY/ABSTRACT Alzheimer’s disease (AD) afflicts millions of Americans, yet no effective treatments exist. Iron has been shown to be involved in key AD pathologic processes, including amyloid and tau aggregation, inflammation, oxidative stress, and cell death mechanisms. Despite this growing evidence, it is challenging to ascertain alterations in iron metabolism in vivo, limiting potential translation to biomarkers and novel therapies. Exosomes are nanometer-sized vesicles shed by cells to transport proteins, nucleic acids, metals, lipids or metabolites. While exosomes reflect cellular processes and can reveal disease-related pathologies in human tissues and biofluids, iron abnormalities in AD exosomes have not yet been investigated. We will address this knowledge gap through state-of-the-art exosome isolation technology combined with advanced iron imaging, protein quantification and next generation sequencing methods. Our goal is to investigate iron dysregulation in exosomes from post- mortem AD brains, in order to unveil AD-specific biomarkers and facilitate the development of novel therapies. The project aims are: (1) To determine whether the quantity, oxidation state, and cellular origin of exosomal iron is altered in AD. Using MRI and synchrotron X-ray microscopy, we will quantify tissue iron content and oxidation state in human AD and control hippocampal specimens. We will then use our novel exosome isolation platform, ExoTIC, to isolate exosomes from regions of high hippocampal iron content in the same specimens. Using antibodies that target cell-surface proteins, we will enrich the isolated exosomes based on their cellular origin (e.g. neurons, microglia, etc.). We will quantify exosomal iron content from each cell type using mass spectrometry (ICP-MS), and measure exosomal iron oxidation state using electron microscopy. Taken together, we will determine whether iron content and oxidation state are altered in Alzheimer’s exosomes compared to controls, in particular in exosomes originating in microglia, the brain’s immune cells. (2) Detect dysregulation of iron-related proteins and RNAs in AD exosomes. Using Western blotting on the enriched exosomes, we will determine whether levels of proteins that play a role in iron metabolism are altered in AD compared to controls. Because exosomes are generally rich in microRNAs that are known to regulate gene expression, we will use RNA-Seq to determine whether exosomal microRNAs regulating these same iron-related proteins are also altered in AD. Machine learning algorithms will enable the creation of an atlas of microRNAs linking iron, iron-related proteins, and neuropathology, which should provide a deeper understanding of AD biology. Characterization of exosome content in the AD brain should result in cell-specific signatures of iron dysregulation associated with neurodegeneration. This approach may elucidate novel aspects of AD biology, lead to novel assays to detect early AD, and facilitate a much-needed future therapy.