Optimization of imaging mass cytometry, a single-cell spatial proteomics technology, for the study of Alzheimer disease

项目摘要 该提案旨在填补应用高维单细胞空间蛋白质组学技术的关键空白- 成像质量细胞仪（IMC）-研究人类大脑中的神经元和神经胶质。IMC是一种新兴的高复杂度 该技术将使用金属同位素标记抗体的免疫组织化学（IHC）染色与 激光烧蚀和基于质谱的检测以产生高维图像。IMC允许 在组织切片上同时检测40-100种蛋白质标记物，并使各种不同的细胞类型成为可能 以空间分辨的单细胞分辨率同时分析。IMC已经应用于癌症， 糖尿病，免疫学和传染病研究，识别功能不同的免疫细胞 与疾病进展、治疗结果和疾病预后的生物标志物相关的亚群。 作为最常见的神经退行性疾病，没有疾病修饰治疗，IMC的应用 阿尔茨海默病（AD）的发病机制和治疗靶点提供了新的见解。 开发有效的治疗方法。这种空间分辨的单细胞分辨率蛋白质组技术至关重要 用于研究AD病理学在空间背景下对不同细胞类型的影响。细胞外的β- 淀粉样蛋白（Aβ）斑块和细胞内神经纤维缠结（NFT）是AD病理学的标志。Aβ 斑块与AD中的选择性神经元死亡有关，并且被活化的星形胶质细胞包围， 小胶质细胞NFT的负荷和位置与神经元丢失、疾病严重程度和临床病程相关。 然而，与细胞和基因表达相关的AD病理学的分子机制， 人类大脑中的变化和神经退行性变仍然不清楚，这严重阻碍了人类大脑的发育。 制定有效的治疗策略。将IMC应用于AD研究的主要挑战是缺乏 一个精心设计的，经过验证的高质量的抗体面板适用于脑组织。这一提议将填补这一空白 通过开发一个37抗体面板，用于使用IMC分析速冻人脑组织， 包括识别中枢神经系统中所有主要细胞类型的标志物和AD的生物标志物 病理学在目标1中，我们将设计和优化37抗体组的免疫检测条件， 用于死后人脑组织切片，并将该技术应用于AD的额叶皮质组织， 匹配的对照病例。在目标2中，我们将开发和优化用于脑组织特异性的计算管道。 IMC数据分析。该技术可用于获取脑的全面空间信息 微环境和神经胶质细胞和神经元之间的细胞-细胞相互作用，这将促进我们的理解 Aβ斑块和NFT的空间分布如何影响胶质细胞活化和神经元死亡。这种优化的 该方法将为神经科学界提供一个强大的IMC神经成像工具，用于广泛的 研究领域，如老化，神经退化和大脑发育。

Project Summary This proposal aims to fill a critical gap in applying high-dimensional single-cell spatial proteomics technology - imaging mass cytometry (IMC) - to study neurons and glia in the human brains. IMC is an emerging high-plex technology which combines immunohistochemistry (IHC) staining using metal isotope-labeled antibodies with laser ablation and mass-spectrometry-based detection to produce high-dimensional images. IMC allows the detection of 40-100 protein markers simultaneously on tissue sections and enables a variety of distinct cell types to be analyzed concurrently at a spatially-resolved single-cell resolution. IMC has been applied to cancer, diabetes, immunology, and infectious disease research, identifying functionally distinct immune cell subpopulations associated with disease progression, treatment outcomes, and biomarkers for disease prognosis. As the most common neurodegenerative disease without a disease-modifying treatment, the application of IMC to Alzheimer disease (AD) could provide novel insights into the disease pathogenesis and targets for the development of effective therapeutic. This spatially-resolved single-cell resolution proteomic technology is critical for investigating the impact of AD pathology on different cell types in a spatial context. The extracellular beta- amyloid (Aβ) plaques and the intracellular neurofibrillary tangles (NFT) are hallmarks of AD pathology. Aβ plaques have been linked to selective neuronal death in AD and are surrounded by activated astrocytes and microglia. The burden and location of NFT correlate with neuronal loss, disease severity, and clinical course. However, the molecular mechanisms underlying AD pathologies in relationship to cellular and gene expression changes and neurodegeneration in the human brain remain unclear, which significantly hampers the development of effective treatment strategies. The main challenge of applying IMC to AD research is the lack of a well-designed, validated high-quality antibody panel suited for the brain tissue. This proposal will fill this gap by developing a 37-antibody panel for the analysis of snap-frozen human brain tissues using IMC which will include both the markers to identify all the major cell types in the central nervous system and biomarkers of AD pathologies. In Aim 1, we will design and optimize the immunodetection conditions for a 37-antibody panel for use on postmortem human brain tissue sections and apply the technology to the frontal cortex tissues of AD and matched control cases. In Aim 2, we will develop and optimize a computational pipeline for brain tissue-specific IMC data analysis. This technology could be used to obtain comprehensive spatial information of brain microenvironment and cell-cell interactions among the glia and neurons, which will advance our understanding of how the spatial distribution of Aβ plaques and NFTs affect glial activation and neuronal death. This optimized methodology will provide the neuroscience community a powerful IMC neuroimaging tool for a broad range of research fields such as aging, neurodegeneration, and brain development.