Project 2: Particle and brain mapping of CSF proteins using elemental reporters with mass spectrometry

摘要 阿尔茨海默病（AD）是对老年人健康的主要威胁，并且是迫在眉睫的公共卫生灾难。 只有通过研究，才能找到诊断和治疗方面有希望的解决方案。项目2建议 获得生物学和医学的见解，在该中心发现的蛋白质，通过多重映射到特定的 CSF细胞外囊泡（EV）和脂蛋白（LP），以及特定细胞和亚细胞结构中的特异性 大脑的区域。 CSF中的蛋白质仍然是AD的最佳生物标志物。CSF蛋白以各种形式存在：游离于 溶液，对一类独特的LP，以及对包括外泌体、微泡和凋亡小体的EV。LP 和EV具有不同的生产机制和生物学功能。定位与相对 将在项目1中发现的感兴趣的蛋白质定量到每类CSF颗粒将提供强有力的 深入了解细胞起源、细胞生物学和潜在医学意义。 通过仔细绘制人脑中的蛋白质，可以获得进一步的生物学和医学见解。的确， 多路成像的力量在于它能够揭示共定位或互斥性，并推断 调节作用，并获得机械的洞察力。例如，我们对生物角色的看法非常不同， 蛋白质的表达限制在突触与线粒体，谷氨酸能与GABA能神经元， 或海马锥体神经元与纹状体中型棘神经元。在病理状态下， 标志性病理结构（主要蛋白质）突出了损伤和对损伤的反应的途径：老年 斑块（淀粉样β肽）、神经退行性变（成对螺旋状的β-tau蛋白）、路易体和 神经突（磷酸-α-突触核蛋白）和磷酸-TDP-43内含物。 我们将检验CSF颗粒以及亚细胞、细胞和细胞内的多重分析是否能用于诊断CSF颗粒的假设。 区域绘图将为从头发现的CSF蛋白提供关键的生物学和医学见解 项目1中CSF的蛋白质组学分析。使用Flow将相同的探针应用于CSF LP和EV 细胞计数，并使用多路离子束成像（MIBI）的脑区域。

Abstract Alzheimer's disease (AD) is a major threat to health of older individuals and is a looming public health disaster. Promised solutions in diagnostics and therapeutics will come only through research. Project 2 proposes to gain biological and medical insights into the proteins discovered in the Center by multiplex mapping to specific CSF extracellular vesicles (EV) and lipoproteins (LP), and to specific cells and subcellular structures in specific regions of brain. Proteins in CSF remain the best performing biomarkers for AD. CSF proteins exist in various forms: free in solution, on a unique class of LP, and on EV that include exosomes, microvesicles, and apoptotic bodies. LP and EV have varying mechanisms of production and biological functions. Localization and relative quantification of proteins of interest discovered in Project 1 to each class of CSF particles will provide powerful insight into cell of origin, cellular biology, and potential medical meaning. Further biological and medical insights can be gained by carefully mapping proteins in human brain. Indeed, the power of multiplexed imaging is its ability to reveal co-localization or mutual exclusivity, and to infer regulatory roles and gain mechanistic insight. For example, we think very differently about the biological roles of proteins restricted in their expression to synapses vs. mitochondria, glutamatergic vs. GABA-ergic neurons, or hippocampal pyramidal neurons vs. striatal medium spiny neurons. In pathologic states, co-localization with hallmark pathologic structures (major protein) highlights pathways of injury and response to injury: senile plaques (amyloid beta peptides), neurofibrillary degeneration (paired helical filament-tau), Lewy bodies and neurites (phospho-alpha-synuclein), and phospho-TDP-43 inclusions. We will test the hypothesis that multiplex analysis of CSF particles as well as subcellular, cellular, and regional mapping will provide key biological and medical insights into CSF proteins discovered by de novo proteomic analysis of CSF in Project 1. The same probes will be applied to CSF LP and EV using flow cytometry, and to brain regions using Multiplexed Ion Beam Imaging (MIBI).