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. Bay?r.
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.
Remarkable advancements of mass spectrometry with regards to small molecules, including lipids, have lead to new concepts about their signaling role, particularly in the coordination of sophisticated interconnectivity and functions of the normal brain and their aberrant responses in the injured brain. However we still do not know in which cells these signaling events are occurring as there are no probes to visualize lipids at the cellular level using even the most contemporary microscopy techniques available today. The goal of this application is to develop, optimize and apply an entirely new and revolutionizing technology ? gas cluster ion beam secondary ion mass spectrometry (CGIB-SIMS) ? for high sub-micron resolution for spatial and temporal mapping of diverse molecular species of phospholipids in cells and subcellular membranes superimposing them onto neuropathology of the injured brain. This new approach ? biochemical microscopy of lipids ? will provide so urgently required but so far unavailable information that is critical for the design and development of targeted cell-specific therapies and evaluation of their efficacy in acute brain injury. We will test novel targeted therapies against cell death for their neuroprotective efficacy after head injury using these approaches.
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