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

• 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
• 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

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.

项目摘要/摘要 大脑功能需要大量的代谢能量，通常是简短的局部爆发。每个脑细胞的能力 响应这种能源需求的代谢机制对于直接功能特性至关重要 大脑信号传导和大脑的长期健康。尽管核心代谢途径由 所有类型的脑细胞，我们假设不同的脑细胞类型可能会强调不同的代谢 响应急性能源需求的组件。例如，神经元和星形胶质细胞可以发挥作用 完全代谢角色；几乎不断发射的神经元，或以非常高的速度发射，可能 管理其新陈代谢与通常静止神经元不同。新陈代谢功能障碍可能导致 疾病和神经变性，细胞类型之间的代谢差异可能是细胞的基础 神经退行性疾病中看到的脑细胞的类型特异性漏洞。 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，我们将使用神经元刺激对小鼠的急性脑切片进行物理实验， 13C代谢标记和代谢抑制剂。然后，我们将使用质谱成像（MSI） 定量地绘制这些大脑切片的薄部分中众多代谢产物的水平。快速热力 刺激或应用后的特定时间保存（闪光加热和冷冻） 13C标记的代谢物使我们能够测量代谢变化的良好时光过程，成像能力 允许我们从特定细胞类型中获得代谢测量。齿状颗粒细胞（DGC）代谢 行为将通过海马紧凑型颗粒细胞层的MSI分离。来自 将使用细胞类型特异 签名，基于与已建立抗体标记的对应关系。 我们将使用这些方法来构造这些单个单元类型如何使用其核心代谢的丰富图片 途径（糖酵解，五磷酸五磷酸五磷酸途径，TCA循环），无论是基线还是动态地响应于 神经元刺激。我们将测试特定的假设，即在DGCS中，神经元糖酵解在 刺激，然后磷酸五磷酸盐途径随后参与。使用燃料分子的实验 具有不同稳定的同位素标签将揭示神经元和星形胶质细胞如何灵活地利用能量的混合物 来源。通过将代谢物水平的数据与有关单个代谢途径活性的数据结合在一起，我们 不仅可以学习代谢变化是什么，而且还可以学习沿哪种键的途径的位置 发生调节性变化。我们将检验以下假设，即DGC，星形胶质细胞和快速刺激性中间神经元 在响应神经元刺激的响应中，使用其核心代谢途径。 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 对于未来的工作，关于新陈代谢如何在衰老或神经退行性疾病中出现问题。