Ex vivo immunocapture and functional characterization of cell-type-specific mitochondria using MitoTag mice.

Mitochondria are key bioenergetic organelles involved in many biosynthetic and signaling pathways. However, their differential contribution to specific functions of cells within complex tissues is difficult to dissect with current methods. The present protocol addresses this need by enabling the ex vivo immunocapture of cell-type-specific mitochondria directly from their tissue context through a MitoTag reporter mouse. While other available methods were developed for bulk mitochondria isolation or more abundant cell-type-specific mitochondria, this protocol was optimized for the selective isolation of functional mitochondria from medium-to-low-abundant cell types in a heterogeneous tissue, such as the central nervous system. The protocol has three major parts: First, mitochondria of a cell type of interest are tagged via an outer mitochondrial membrane eGFP by crossing MitoTag mice to a cell-type-specific Cre-driver line or by delivery of viral vectors for Cre expression. Second, homogenates are prepared from relevant tissues by nitrogen cavitation, from which tagged organelles are immunocaptured using magnetic microbeads. Third, immunocaptured mitochondria are used for downstream assays, e.g., to probe respiratory capacity or calcium handling, revealing cell-type-specific mitochondrial diversity in molecular composition and function. The MitoTag approach enables the identification of marker proteins to label cell-type-specific organelle populations in situ, elucidates cell-type-enriched mitochondrial metabolic and signaling pathways, and reveals functional mitochondrial diversity between adjacent cell types in complex tissues, such as the brain. Apart from establishing the mouse colony (6-8 weeks without import), the immunocapture protocol takes 2 h and functional assays require 1-2 h.

Ketogenic diet uncovers differential metabolic plasticity of brain cells.

To maintain homeostasis, the body, including the brain, reprograms its metabolism in response to altered nutrition or disease. However, the consequences of these challenges for the energy metabolism of the different brain cell types remain unknown. Here, we generated a proteome atlas of the major central nervous system (CNS) cell types from young and adult mice, after feeding the therapeutically relevant low-carbohydrate, high-fat ketogenic diet (KD) and during neuroinflammation. Under steady-state conditions, CNS cell types prefer distinct modes of energy metabolism. Unexpectedly, the comparison with KD revealed distinct cell type-specific strategies to manage the altered availability of energy metabolites. Astrocytes and neurons but not oligodendrocytes demonstrated metabolic plasticity. Moreover, inflammatory demyelinating disease changed the neuronal metabolic signature in a similar direction as KD. Together, these findings highlight the importance of the metabolic cross-talk between CNS cells and between the periphery and the brain to manage altered nutrition and neurological disease.