Wednesday, January 15, 2025
1/15/2025
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.
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