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

摘要 阿尔茨海默病(AD)是对老年人健康的一大威胁，也是一场迫在眉睫的公共卫生灾难。 在诊断和治疗方面承诺的解决方案只有通过研究才能实现。项目2建议 通过对特定蛋白质的多重作图，获得对中心发现的蛋白质的生物学和医学洞察力 脑脊液细胞外小泡(EV)和脂蛋白(LP)，以及特定的细胞和亚细胞结构在特定的 大脑的各个区域。 脑脊液中的蛋白质仍然是AD表现最好的生物标志物。脑脊液蛋白以各种形式存在：游离存在 包括外切体、微泡和凋亡小体在内的EV。低压 和EV具有不同的生产机制和生物学功能。本地化与相对化 将项目1中发现的感兴趣蛋白质定量到每一类脑脊液颗粒将提供强大的 深入了解细胞起源、细胞生物学和潜在的医学意义。 通过仔细绘制人类大脑中的蛋白质图，可以获得进一步的生物学和医学见解。的确， 多路复用成像的能力在于它能够揭示共同定位或相互排斥，并能够推断 监管角色和获得机械性的洞察力。例如，我们对生物角色的看法非常不同 仅限于突触和线粒体、谷氨酸能和GABA能神经元的蛋白质， 或海马区锥体神经元与纹状体中棘神经元。在病理状态下，与 标志性病理结构(主要蛋白质)突出显示损伤的途径和对损伤的反应：老年人 斑块(淀粉样β多肽)、神经纤维变性(成对螺旋微丝-tau)、路易小体和 突起(磷酸-α-突触核蛋白)和磷酸-TDP-43包裹体。 我们将检验这一假设，即对脑脊液颗粒以及亚细胞、细胞和 区域图谱将为新发现的脑脊液蛋白提供关键的生物学和医学见解 项目1的脑脊液蛋白质组学分析。相同的探针将用于脑脊液LP和EV使用FLOW 使用多路离子束成像(MIBI)对脑区进行检测。

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).