Dynamics of cellular brain metabolism using mass spectrometry imaging

使用质谱成像研究细胞脑代谢动力学

基本信息

批准号：
10418219
负责人：
Nathalie YR Agar
金额：
$ 44.99万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10418219
关键词：
Action Potentials Acute Address Aging Antibodies Antioxidants Astrocytes Behavior Biosensor Brain Cell Culture Techniques Cells Citric Acid Cycle Clustered Regularly Interspaced Short Palindromic Repeats Complement Complex Data Disease Energy Metabolism Energy Supply Energy-Generating Resources Exhibits Fatty Acids Fire - disasters Foundations Freezing Functional disorder Future Glucose Glutamine Glycolysis Habits Heating Hippocampus (Brain)Individual Interneurons Ketone Bodies Knowledge Label Lead Learning Lipids Maps Measurement Measures Mediating Metabolic Metabolic Pathway Metabolism Methods Microtomy Mus NADP Nerve Degeneration Neurodegenerative Disorders Neurons Oxidants Oxidation-Reduction Oxygen Parvalbumins Pathway interactions Pentosephosphate Pathway Pharmacology Physiological Play Positioning Attribute Positron-Emission Tomography Process Property Proteins Regulation Role Signal Transduction Slice Stable Isotope Labeling Stress Technology Testing Therapeutic Time Tissues Work base brain cell brain health brain metabolism brain tissue cell type experimental study flexibility gene product granule cell imaging capabilities imaging modality in vivo inhibitor knock-down mass spectrometric imaging metabolic abnormality assessment metabolomics neuronal metabolism neuropathology preservation public health relevance relating to nervous system response sensor

项目摘要

PROJECT SUMMARY/ABSTRACT Brain function demands a lot of metabolic energy, often in brief, local bursts. The ability of each brain cell’s metabolic machinery to respond to this energy demand is crucial both for the immediate functional properties of brain signaling and for the long-term health of the brain. Although the core metabolic pathways are shared by all types of brain cells, we hypothesize that different brain cell types are likely to emphasize different metabolic components in response to acute energy demand. For instance, neurons and astrocytes are thought to play complementary metabolic roles; and neurons that fire nearly constantly, or episodically at very high rates, may manage their metabolism differently from typically quiescent neurons. Dysfunction in metabolism can lead to disease and neurodegeneration, and the metabolic differences between cell types may underlie the very cell- type-specific vulnerabilities of brain cells seen in neurodegenerative diseases. To study the distinctive, dynamic metabolic responses of specific cell types in intact tissue, rather than cell culture, we will perform physiological experiments on acute brain slices from mice, using neuronal stimulation, 13C metabolic labeling, and metabolic inhibitors. We will then use mass spectrometry imaging (MSI) to quantitatively map the levels of numerous metabolites in thin sections from those brain slices. Fast thermal preservation (flash heating and freezing) of the brain slices at specific times after stimulation or application of 13C-labeled metabolites allows us to measure a fine time course of metabolic changes, and the imaging capability allows us to obtain metabolic measurements from specific cell types. Dentate granule cell (DGC) metabolic behavior will be isolated by MSI of the compact granule cell layer of the hippocampus; the metabolic signals from single astrocytes and fast-spiking parvalbumin-positive interneurons will be isolated using cell-type specific signatures, based on correspondence with labeling by established antibodies. We will use these methods to construct a rich picture of how these individual cell types use their core metabolic pathways (glycolysis, pentose phosphate pathway, TCA cycle), both at baseline and dynamically in response to neuronal stimulation. We will test the specific hypotheses that in DGCs, neuronal glycolysis is upregulated after stimulation, and that the pentose phosphate pathway then becomes engaged. Experiments using fuel molecules with different stable isotope labels will reveal how neurons and astrocytes flexibly utilize a mixture of energy sources. By combining data on metabolite levels with data on the activity of individual metabolic pathways, we can learn not only what the metabolic changes are, but also the positions along each pathway at which key regulatory changes occur. And we will test the hypothesis that DGCs, astrocytes, and fast-spiking interneurons use their core metabolic pathways distinctively in response to neuronal stimulation. This project will reveal the distinctive metabolism of different cell types in healthy brain tissue and lay a foundation for future work on how metabolism may go awry (as is suspected) in aging or in neurodegenerative disease.

项目概要/摘要大脑功能需要大量的代谢能量，通常是短暂的、局部的爆发。响应这种能量需求的代谢机制对于直接功能特性至关重要尽管核心代谢途径是共享的。所有类型的脑细胞，我们追求不同的脑细胞类型可能强调不同的代谢例如，神经元和星形胶质细胞被认为会发挥作用。互补的代谢作用；几乎持续或间歇性地以非常高的速率放电的神经元可能其新陈代谢的管理方式与典型的静止神经元不同。新陈代谢功能障碍可能会导致。疾病和神经退行性疾病，以及细胞类型之间的代谢差异可能是细胞- 神经退行性疾病中脑细胞的特定类型脆弱性。研究完整组织中特定细胞类型的独特动态代谢反应，而不是细胞培养，我们将使用神经刺激对小鼠的急性脑切片进行生理实验，然后我们将使用质谱成像 (MSI) 来进行 13C 代谢标记和代谢抑制剂。定量绘制这些脑切片薄片中多种代谢物的水平。刺激或应用后特定时间保存（快速加热和冷冻）脑切片 13C 标记的代谢物使我们能够测量代谢变化的精细时间过程，并且成像能力使我们能够获得特定细胞类型的代谢测量结果。行为将通过海马致密颗粒细胞层的 MSI 来分离；将使用细胞类型特异性分离单个星形胶质细胞和快速尖峰小清蛋白阳性中间神经元签名，基于与已建立的抗体标记的对应关系。我们将使用这些方法来构建一幅丰富的图景，展示这些个体细胞类型如何利用其核心代谢途径（糖酵解、磷酸戊糖途径、TCA 循环），无论是在基线还是动态响应我们将测试 DGC 中神经元糖酵解在刺激后上调的具体假设。刺激，然后磷酸戊糖途径开始使用燃料分子进行实验。不同的稳定同位素标记将揭示神经元和星形胶质细胞如何灵活地利用能量混合物通过将代谢水平数据与个体代谢途径活动数据相结合，我们不仅可以了解代谢变化是什么，还可以了解每个途径的关键位置我们将检验 DGC、星形胶质细胞和快速尖峰中间神经元的假设。独特地利用其核心代谢途径来响应神经元刺激。该项目将揭示健康脑组织中不同细胞类型的独特代谢，并奠定基础未来研究新陈代谢如何在衰老或神经退行性疾病中出错（正如所怀疑的那样）。