Nrf2 affects mitochondrial NADH levels in neurons and astrocytes and NADPH levels in astrocytes (A) Scheme showing main pathways of cellular glucose metabolism: glycolysis resulting in NADH production in the mitochondria and pentose phosphate pathway (PPP), resulting in NADPH production. (B) Representative traces showing mitochondrial NADH homeostasis, as measured by its autofluorescence, in astrocytes. Uncoupler FCCP maximizes respiration, leading to consumption of all mitochondrial NADH. Remaining fluorescence at this stage is attributed to NADPH. Subsequent application of NaCN inhibits respiration leading to mitochondrial NADH accumulation. Redox index is the relative NADH level in basal conditions of the total mitochondrial NADH pool. (C-H) Quantification of: redox index (C,F), mitochondrial NADH pool (D,G), and relative NADPH pool (E,H) as explained in (B) in WT, Nrf2KO and Keap1 KD individual neurons and astrocytes. Basal (25 mM glucose), low glucose (12 h incubation in 1 mM glucose) and orthovanadate (Basal plus OV 30 μM for 12 h). Mitochondrial NADH and NADPH pools are expressed as a % of basal WT. Number of cells analysed is shown in brackets. Non-parametric Kruskal-Wallis ANOVA with post-hoc Dunn's test for each group, *p < 0.05, **p < 0.01, ***p < 0.0001.