Lipid imaging in Traumatic Brain Injury by high resolution GCIB-secondary ion mass spectrometry

自从三百多年前显微镜的发明和对细胞的初步观察以来，细胞 生物学在细胞内细胞器的详细结构和功能表征方面取得了胜利 大分子复合物。认识到专门的双功能分子，脂质，可以形成 膜结构的水界面引起了人们对这组细胞内化合物的关注。 广泛的生化研究发现脂质存在巨大的多样性，无法适应 它们作为膜构件的作用的简单概念。事实上，许多信号功能 已经发现了不同的脂质分子，包括膜脂。尽管非常成功 无数脂质的生化特征分析工作，精确的细胞内拓扑结构 脂质的单个分子种类在其信号传导功能方面尚未确定。的 造成这种情况的主要原因是缺乏足够的技术来对小脂质进行高分辨率成像 分子。气体团簇离子束二次离子质谱技术的最新进展 （GCIB-SIMS）首次填补了细胞生物学基础知识的空白，并开发了一种 新型显微镜——脂质生化显微镜——将创建个体的细胞内图谱 脂质及其生命必需品在生物膜中的不对称分布。本次目标的实现 创新和范式转变工作将基于高度跨学科的方法和领导力 三个团队在SIMS（宾夕法尼亚大学）各自分析/物理化学领域的地位 州立大学，N. Winograd），脂质生物化学/生物学（匹兹堡大学，V.E. Kagan），以及 创伤性脑损伤 (TBI)（匹兹堡大学 H. Bayır。目标 1 将采用高分辨率 GCIB-SIMS 探索神经元、胶质细胞和小胶质细胞中的分子形态并构建 CL 和 PE 的细胞特异性图谱 正常小鼠大脑不同解剖区域的细胞。目标 2 将识别 TBI 诱导的分子 使用神经元、胶质细胞和小胶质细胞中心磷脂 (CL) 和磷脂酰乙醇胺 (PE) 的变化 小鼠控制皮质冲击 (CCI) 模型中的 GCIB-SIMS。我们将进一步确定 TBI 引起的变化 与凋亡或铁死亡相关的单个 CL 和 PE 物种的亚细胞分布 各个单元中的程序。我们对线粒体 CL 中促凋亡的变化特别感兴趣 和PE中的促铁死亡变化。我们还将检查从难治性 TBI 患者身上取出的脑组织 颅内高压和脑库对照组织。目标 3 将确定 GCIB-SIMS 成像的实用性 评估选定的抗凋亡和抗铁死亡小分子调节剂的有效性 防止 TBI 后 CL 和 PE 分子形态的细胞特异性变化。拟议的研究将解码 细胞和组织中各种脂质分子的拓扑特征及其在细胞和组织中的作用 健康和疾病中的信号功能。

Since the invention of microscopy and the initial observation of cells more than three hundred years ago, cell biology has been triumphant in detailed structural and functional characterization of intracellular organelles and macromolecular complexes. The realization that specialized bi-functional molecules, lipids, can form the aqueous interfaces of membrane structures has attracted attention to this group of intracellular compounds. Extensive biochemical studies discovered a huge diversification of lipids that could not be accommodated within a simple concept of their role as membrane building blocks. Indeed, numerous signaling functions of different lipid molecules, including membrane lipids, have been discovered. In spite of the very successful analytical work in biochemical characterization of the countless lipids, the exact intracellular topography of individual molecular species of lipids in the context of their signaling functions has not been established. The major reason for this was the lack of adequate technologies for high resolution imaging of small lipid molecules. The most recent developments of Gas Cluster Ion Beams Secondary Ion Mass Spectrometry (GCIB-SIMS) allows, for the first time, to fill this gap of fundamental knowledge in cell biology and develop a new type of microscopy – biochemical microscopy of lipids – that will create intracellular maps of individual lipids and their essential for life asymmetric distribution in biomembranes. Achievement of the goals of this innovative and paradigm shifting work will be based on highly interdisciplinary approaches and the leadership position of the three teams in their respective fields of analytical/physical chemistry of SIMS (at Pennsylvania State University, N. Winograd), lipid biochemistry/biology (at the University of Pittsburgh, V.E. Kagan), and traumatic brain injury (TBI) (at University of Pittsburgh, H. Bayır. Aim 1 will employ high-resolution GCIB-SIMS to explore molecular speciation and construct cell-specific maps of CL and PE in neuronal, glial, and microglial cells in different anatomical regions of normal mouse brain. Aim 2 will identify TBI induced molecular alterations in cardiolipin (CL) and phosphatidylethanolamine (PE) in neuronal, glial, and microglial cells using GCIB-SIMS in mouse controlled cortical impact (CCI) model. We will further identify TBI induced changes in subcellular distribution of individual CL and PE species related to the execution of apoptotic or ferroptotic programs in the respective cells. We will be particularly interested in pro-apoptotic changes in mitochondrial CL and pro-ferroptotic changes in PE. We will also examine brain tissue removed from TBI patients with refractory intracranial hypertension and brain-bank control tissue. Aim 3 will determine the utility of GCIB-SIMS imaging in assessing the effectiveness of select anti-apoptotic and anti-ferroptotic small molecule regulators in preventing cell-specific changes in CL and PE molecular speciation after TBI. Proposed studies will decode specific features of topography of individual types of lipid molecules in cells and tissues and their role in signaling functions in health and disease.