Fig 2: GDAP1 knockdown uncouples mitochondrial respiration from ATP generation and shifts metabolism towards glutaminolysis.a Mitochondrial oxygen consumption of intact cells in regular growth medium measured by high-resolution respirometry and corrected for ROX. ETS, electron transfer system; L, leak respiration; R, routine respiration; E, ETS capacity; ROX, residual oxygen consumption; netR, oxygen consumption minus ROX. Calculation of flux control ratios. b Comparison of cytosolic and mitochondrial ATP content using the genetically encoded ATP sensor BTeam. Calculation of BTeam YFP/NLuc emission ratios under basal conditions revealed reduced mitochondrial ATP levels. c Immunoblot quantification of proteins involved in glycolysis in Ctrl and KD cells shows an increase in lactate dehydrogenase A (LDHA) and glutamate dehydrogenase 1 (GLUD1) levels. HK, Hexokinase; G6PD, glucose-6-phosphate dehydrogenase; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; PKM1, pyruvate kinase M1; PDH, pyruvate dehydrogenase; pPDK1, phospho-pyruvate dehydrogenase kinase. Actin expression served as loading control. d Diminished glutamate and glutamine levels determined by a luminescence-based assay. e Comparison of cytosolic ATP content using the genetically encoded ATP sensor BTeam. BTeam YFP/NLuc emission ratios after treatment with 25 µM 2-deoxyglucose reveals increased non-glucose dependent ATP generation capacity in KD cells. f Automated high-content confocal microscopy analysis of BODIPY-stained fatty acids demonstrating less lipid droplets in KD cells identified by Höchst staining of nuclei. Lipid droplets close to mitochondria were identified by MitoTracker staining. g ETS capacity in the presence of palmitate or BSA as substrates measured by high-resolution respirometry. h Schematic illustration of metabolic changes observed in GDAP1 KD cells. Upregulation is shown in blue and bold lines; downregulation in magenta and dashed lines. ME, malic enzyme 1; PDC, pyruvate dehydrogenase complex; PKM1/2, pyruvate kinase M1/2; OAA, oxaloacetate. Data in (a) and (a') are from 12 independent experiments performed in duplicate. Data in (b) are from 4 independent experiments performed in triplicates. Data in (e) are from 6 independent experiments performed in triplicates. Data in (f) were from >10,000 cells in total and were analyzed in 3 independent experiments performed in triplicates. Statistical variation is shown as Tukey’s boxplots in (a) and (b), XY graph in (c), scatter plots in (d) and (f), mean ± SEM in (e). Significance was calculated using the student’s t test in (a), 2-way ANOVA in b, the non-parametric Kruskal–Wallis test in (c), the Mann–Whitney test in (d–g), *p < 0.05, **p < 0.001, ***p < 0.0001.

Fig 3: Metabolic reprogramming in Lal–/– CD11c+ cells.(A and B) Gene expression of glycolysis (A) and citrate cycle (B) across cell clusters in t-SNE plots of CD11c+ cells from Lal–/– versus Lal+/+ mice. (C) Statistical analysis of ROS MFI in Lal–/– versus Lal+/+ CD11c+ cells by flow cytometry. (D) Expression of gene response to ROS across cell clusters in t-SNE plots of CD11c+ cells from Lal–/– versus Lal+/+ mice. (E) Extracellular acidification rate (ECAR) of glycolysis and oxygen consumption rate (OCR) in mitochondrial respiration in Lal–/– versus Lal+/+ CD11c+ cells. (F) ATP production in mitochondrial respiration in Lal–/– versus Lal+/+ CD11c+ cells. (G) MFI of G6PD, PDH, LDH, and GLUD expression in Lal–/– versus Lal+/+ CD11c+ cells by flow cytometry. (H) Percentage of CD11c+ cells in the blood and percentage of PD-L1+ cells in CD11c+ blood cells after CPI-613 treatment by flow cytometry analysis. (I) CPI-613–pretreated CD11c+ cells were cocultured with CFSE-labeled Lal+/+ CD4+ T cells for T cell proliferation assay. (J) CPI-613–pretreated CD11c+ cells (2 × 105) were coinjected with B16 melanoma cells (2 × 105) into the flank region of Lal+/+ recipient mice for tumor growth assay. Data are expressed as mean ± SD. Experiments were independently repeated, n = 4 for C, G, and I, n = 6–8 for E and F, n = 7 for H, n = 12 for J. *P < 0.05, **P < 0.01, unpaired Student’s t test for C, F, and G, 2-way ANOVA for H and I, 1-way ANOVA for J.

Fig 4: MPC inhibition affects glutamine–to–TCA cycle flux in DLBCL cells.(A) Growth assay of OxPhos- and BCR-DLBCL cells cultured in suspension in media supplemented with 2, 1, 0.75, 0.5, 0.25, or 0 mM glutamine ± the MPC inhibitor UK-5099. Cell concentration is the mean of n = 3 independent biological experiments, ± SD. (B) Growth assay of OxPhos- and BCR-DLBCL cells cultured in suspension and treated with either vehicle, GLS inhibitor CB-839, or CB-839 with dimethyl-a-ketoglutarate (dmKG). Cell concentration is the mean of n = 3 independent biological experiments, ± SD. (C) Schematic of l-[U-13C]-glutamine tracing. GOT2, mitochondrial aspartate aminotransferase; GDH, glutamate dehydrogenase. (D) Quantification of the isotopologue abundances of M + 5 citrate, M + 4 succinate, M + 4 malate, M + 4 citrate, M + 2 succinate, and M + 2 malate in DLBCL cells cultured with l-[U-13C]-glutamine ± the MPC inhibitor UK-5099 for 30 min. Isotopologue abundance is the mean of n = 3 independent biological experiments, ± SD. (E) Quantification of the isotopologue abundances of M + 5 citrate, M + 4 succinate, M + 4 malate, M + 4 citrate, M + 2 succinate, and M + 2 malate in DLBCL cells cultured with l-[U-13C]-glutamine ± the MPC inhibitor UK-5099 for 2 hours. Isotopologue abundance is the mean of n = 3 independent biological experiments, ± SD. (F) Quantification of the isotopologue abundances of M + 0 and M + 3 alanine and M + 0 and M + 3 lactate in the medium collected from DLBCLs grown with d-[U-13C]-glucose ± the MPC inhibitor UK-5099 for 4 hours. Isotopologue abundance is the mean of n = 3 independent biological experiments, ± SD. Vehicle: Dimethyl sulfoxide (DMSO). ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test.

Fig 5: Patient-derived cells are similarly characterized by an anaplerotic state and reduced mitochondrial Ca2+ levels.a Differentiation protocol to obtain motoneurons (MN) from induced-pluripotent stem cells (iPSCs) and (b) confirmatory immunocytochemistry by staining against dendrite marker MAP2 and motoneuronal neurofilament Smi32. c Immunoblot of total MN cell lysates for validation of GDAP1 expression, actin served as loading control, size is indicated. d Automated high-content microscopy analysis of MitoTracker-stained mitochondria revealed an increased total mitochondrial mass per well. e Immunoblot of total MN cell lysates demonstrated increased glutamate dehydrogenase (GLUD1, arrowhead) in patient-derived MNs, actin served as loading control. f Reduced glutamate levels in CMT4A patient-derived MN determined by a luminescence-based assay indicating increased glutamine consumption compared to Ctrl. g Automated high-content microscopy analysis of BODIPY-positive spots normalized to the nuclei area in MN revealed a reduced number of lipid droplets in CMT4A patient-derived cells. h Reduced relative mt[Ca2+] levels of NPCs determined by confocal microscopy with Rhod2-AM and mito-CEPIA3. i Immunoblots and quantification demonstrating increased phosphorylation of PDH E1 serine 293, normalized to total PDH E1 levels and control cells. Data in (d–g) were from 3 independent differentiations and data in (d, g) were obtained from n > 40,000 cells in quadruplicate, experiments in (f) were performed in duplicate. Data in (h) were from 20–22 Rhod2-AM-stained cells and 22–33 cells mitoCEPIA3 transfected from three independent experiments. Data in (i) were from 6 different passages. Data points corresponding to the example blots are highlighted. Statistical variation is shown as Tukey boxplots in (d, e) or scatter plots in (i) with mean ± SEM indicated. Significance was calculated using the non-parametric Kruskal–Wallis test, *p < 0.05, **p < 0.001, ***p < 0.0001.