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

项目摘要 这一提议旨在填补应用高维单细胞空间蛋白质组技术的一个关键空白-- 成像质量细胞术(IMC)-研究人脑中的神经元和神经胶质细胞。IMC是一种新兴的高端企业 将使用金属同位素标记抗体的免疫组织化学(IHC)染色与 激光消融和基于质谱学的检测，以产生高维图像。IMC允许 在组织切片上同时检测40-100个蛋白质标记物，使各种不同的细胞类型成为可能 以空间分辨的单胞分辨率同时进行分析。IMC已被应用于癌症， 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，没有疾病的治疗，IMC的应用 对于阿尔茨海默病(AD)的研究可以为疾病的发病机制和靶点提供新的见解 开发有效的治疗方法。这种空间分辨的单细胞蛋白质组技术是至关重要的 用于研究AD病理对空间背景下不同细胞类型的影响。细胞外的β- 淀粉样蛋白(Aβ)斑块和细胞内神经原纤维缠结(NFT)是AD病理的特征。Aβ 斑块与AD的选择性神经元死亡有关，并被激活的星形胶质细胞和 小胶质细胞。NFT的负担和位置与神经元丢失、疾病严重程度和临床病程相关。 然而，AD病理基础的分子机制与细胞和基因表达有关 人脑的变化和神经退化仍不清楚，这显著阻碍了 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、适合脑组织的高质量抗体小组。这项提议将填补这一空白。 通过开发一个包含37个抗体的小组，用于使用IMC分析快速冷冻的人脑组织，这将 包括用于识别中枢神经系统中所有主要细胞类型的标记和AD的生物标记 病理学。在目标1中，我们将设计和优化37个抗体小组的免疫检测条件 在死后人脑组织切片上的应用以及在AD和AD的额叶皮质组织中的应用 配对的对照病例。在目标2中，我们将开发和优化用于特定脑组织的计算管道 IMC数据分析。该技术可用于获取大脑的全面空间信息 胶质细胞和神经元之间的微环境和细胞间的相互作用，这将促进我们的理解 Aβ斑块和NFT的空间分布如何影响神经胶质细胞的激活和神经元死亡。这是经过优化的 方法论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 研究领域包括衰老、神经退行性变和脑发育。

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.