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 生物学在详细的细胞内细胞器和功能的结构和功能表征方面取得了胜利 大分子复合体。认识到特殊的双功能分子，脂类，可以形成 膜结构的水界面引起了人们对这类细胞内化合物的关注。 广泛的生化研究发现，无法适应的脂质种类繁多。 在一个简单的概念中，它们作为膜构建块的作用。事实上，大量的信号传递功能 不同的脂质分子，包括膜脂，已经被发现。尽管非常成功， 在生化表征无数脂类方面的分析工作，准确的细胞内形貌 脂类的个别分子物种在其信号功能的背景下还没有确定。这个 主要原因是缺乏足够的小分子脂类高分辨率成像技术。 分子。气体团簇离子束二次离子质谱仪的最新进展 (GCIB-SIMS)首次允许填补细胞生物学基础知识的这一空白，并开发出一种 新型显微镜--脂质的生化显微镜--将创建个体的细胞内图谱 脂类及其生命必需物质在生物膜中的不对称分布。实现本组织的目标 创新和范式转换工作将基于高度跨学科的方法和领导力 三个小组在各自的SIMS分析/物理化学领域的地位(在宾夕法尼亚州 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(匹兹堡大学H.Bayır.Aim 1将采用高分辨率GCIB-SIMS 探讨神经元、神经胶质细胞和小胶质细胞CL和PE的分子形态并构建细胞特异性图谱 正常小鼠脑内不同解剖区域的细胞。AIM 2将鉴定脑损伤诱导的分子 脑神经细胞、胶质细胞和小胶质细胞心磷脂(CL)和磷脂酰乙醇胺(PE)的变化 GCIB-SIMS在小鼠控制皮质撞击(CCI)模型中的作用。我们将进一步确定TBI导致的变化 与执行细胞凋亡性或上皮性铁有关的单个CL和PE物种的亚细胞分布 在各自的单元中编程。我们将对线粒体CL的促凋亡变化特别感兴趣 PE中的前铁链改变。我们还将检查从患有难治性脑损伤的患者中取出的脑组织。 颅内高压和脑库控制组织。目标3将确定GCIB-SIMS成像的用途 在评估选定的抗细胞凋亡和抗铁上链小分子调节剂在 防止颅脑损伤后CL和PE分子形态的细胞特异性变化。拟议的研究将破译 细胞和组织中不同类型脂类分子的特殊形态特征及其在 信号在健康和疾病中的作用。

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.