Neurons depend on oxidative phosphorylation for energy generation, whereas astrocytes do not, a distinctive feature that is essential for neurotransmission and neuronal survival. However, any link between these metabolic differences and the structural organization of the mitochondrial respiratory chain is unknown. Here, we investigated this issue and found that, in neurons, mitochondrial complex I is predominantly assembled into supercomplexes, whereas in astrocytes the abundance of free complex I is higher. The presence of free complex I in astrocytes correlates with the severalfold higher reactive oxygen species (ROS) production by astrocytes compared with neurons. Using a complexomics approach, we found that the complex I subunit NDUFS1 was more abundant in neurons than in astrocytes. Interestingly, NDUFS1 knockdown in neurons decreased the association of complex I into supercomplexes, leading to impaired oxygen consumption and increased mitochondrial ROS. Conversely, overexpression of NDUFS1 in astrocytes promoted complex I incorporation into supercomplexes, decreasing ROS. Thus, complex I assembly into supercomplexes regulates ROS production and may contribute to the bioenergetic differences between neurons and astrocytes.redox | brain | bioenergetics | lactate | glycolysis T he brain is a metabolically demanding organ (1) that requires tight cooperation between neurons and astrocytes (2). Astrocytes provide crucial metabolic and structural support (3, 4) and are key players in neurotransmission (5-7) and behavior (8). The status of many major redox couples in the brain is also regulated by astrocytes (9), through their high content of antioxidant compounds and enzymes (10) and by the constitutive stabilization of the master antioxidant transcriptional activator, nuclear factor erythroid 2-related factor 2 (Nrf2) (11). Thus, astrocytes are equipped to protect themselves when exposed to excess reactive oxygen species (ROS) (12) and reactive nitrogen species (13,14). Moreover, astrocytes also provide nearby neurons with protective antioxidant precursors through a cell-signaling mechanism involving glutamate receptor activation by neurotransmission (11,15,16). The tight coupling between astrocytes and neurons therefore helps in energy and redox metabolism during normal brain function.Intriguingly, the ATP used by neurons is supplied by oxidative phosphorylation, whereas most energy needs of astrocytes are met by glycolysis (17). In fact, the survival of neurons requires oxidative phosphorylation (18,19). The different energy metabolisms of the two cell types are closely coupled, with astrocytes releasing the glycolytic end product, lactate, which is used by neighboring neurons to drive oxidative phosphorylation (20)(21)(22). As the molecular mechanisms underlying the markedly different modes of ATP production in the two cell types are not understood, we investigated whether the organization of the mitochondrial respiratory chain in brain cells could contribute. Here, we report that the extent of supercomplex f...