Mitochondrial dysfunction associated with TANGO2 deficiency

Abstract

Transport and Golgi Organization protein 2 Homolog (TANGO2)-related disease is an autosomal recessive disorder caused by mutations in the TANGO2 gene. Symptoms typically manifest in early childhood and include developmental delay, stress-induced episodic rhabdomyolysis, and cardiac arrhythmias, along with severe metabolic crises including hypoglycemia, lactic acidosis, and hyperammonemia. Severity varies among and within families. Previous studies have reported contradictory evidence of mitochondrial dysfunction. Since the clinical symptoms and metabolic abnormalities are suggestive of a broad dysfunction of mitochondrial energy metabolism, we undertook a broad examination of mitochondrial bioenergetics in TANGO2 deficient patients utilizing skin fibroblasts derived from three patients exhibiting TANGO2-related disease. Functional studies revealed that TANGO2 protein was present in mitochondrial extracts of control cells but not patient cells. Superoxide production was increased in patient cells, while oxygen consumption rate, particularly under stress, along with relative ATP levels and β-oxidation of oleate were reduced. Our findings suggest that mitochondrial function should be assessed and monitored in all patients with TANGO2 mutation as targeted treatment of the energy dysfunction could improve outcome in this condition.

Figure 1

Presence of TANGO2 protein in mitochondria and loss of mitochondrial function through cellular respiration, ATP production, and fatty acid oxidation. (a) Whole cell extracts from fibroblasts of three patients with TANGO2-related disease and a control were analyzed for the presence of TANGO2 antigen by SDS-PAGE western blotting with anti-TANGO2 and anti-GAPDH antibodies. Twenty-five μg protein was loaded for all. Cropped images shown, full-length blots are presented in Supplementary Fig. S1. (b) Mitochondrial extracts from fibroblasts of three patients with TANGO2 disorder and a control were analyzed for the presence of TANGO2 antigen by SDS-PAGE western blotting with anti-TANGO2, anti-β-actin and anti-Hsp60 antibodies. Thirty μg protein was loaded for all. Cropped images shown, full-length blots are presented in Supplementary Fig. S2. (c) Oxygen Consumption Rate (OCR) data was measured with a Seahorse XFe96 Extracellular Flux Analyzer and presented as basal respiration, maximal respiration, and spare capacity for both conditions: with and without glucose. With glucose, only P1 and P2 show reduced oxygen consumption of all 3 measures relative to control while when under stress, in the no glucose condition, all patients show reduced OCR in all measures. Data presented as pmol/min/μg protein. (d) ATP content monitored using the ATPlite™ bioluminescence assay showing significantly reduced ATP production in all patients relative to control. (e) ATP Production Rate was measured with a Seahorse XFe96 Extracellular Flux Analyzer to calculate distinct ATP production from glycolysis and mitochondrial respiration when incubated with and without glucose. Total ATP production rate was increased in P1 and P3 relative to control in normal conditions. Without glucose, the patients ATP production rates do not differ from control. (f) Tritium release assay performed with tritiated palmitate showing reduced palmitate metabolism through the β-oxidation pathway. p value: ****≤ 0.0001; ***≤ 0.001; **≤ 0.01; *≤ 0.05; ns > 0.05. Error bars represent standard error.

Figure 2

Assessment of mitochondrial damage. (a) Reactive oxygen species (ROS) production measured using MitoSox Red and normalized to mitochondrial mass as measured using MitoTracker Green, with and without glucose. ROS production is high in P1 and P2 relative to control in both conditions. (b) Mitochondrial membrane potential as measured using live-cell imaging of JC-1 dye, showing reduced Red/Green (hyperpolarized/depolarized) fluorescence ratio, and therefore depolarized membrane potential in P1 and P2 relative to control. (c) Transmission electron microscopy images at 10,000X magnification from patients and control, showing no visual differences in the fine structure of patient mitochondria. p value: ****≤ 0.0001; ***≤ 0.001; **≤ 0.01; *≤ 0.05; ns > 0.05. Error bars represent standard error.

Figure 3

Evaluation of mitochondrial dynamics. (a) Immunofluorescent staining of fibroblasts from TANGO2 patients and a control. TOMM20 antigen was visualized with green fluorescently tagged antibodies and analyzed using the Nikon NIS-Elements software for mitochondrial number and volume. Nuclei were visualized with Hoescht-Blue stain. Large scale (3 × 3) images were taken at 60 × magnification. Images presented are cropped for easier visualization. Patients were observed to have significantly increased mitochondrial number and decreased volume per mitochondrion, suggesting more fractured mitochondria relative to control. (b) Digital droplet PCR was used to measure mitochondrial DNA copy number using 2 mitochondrial genes: ND1 and CytB, and 2 nuclear genes: B2M and RPP30. Data is presented as the ratio of mitochondrial DNA copy number:nuclear DNA copy number. (c) Mitochondrial fission and fusion dynamics, as well as the ER-mitochondrial crosstalk axis and relative ER stress were assessed via SDS-PAGE western blotting of whole cell extracts from fibroblasts from patients and control. A variety of antigens known to be involved in these pathways were used. Twenty-five μg protein was loaded for all. Cropped images shown, full-length blots are presented in Supplementary Figs. S3–S6. p value: ****≤ 0.0001; ***≤ 0.001; **≤ 0.01; *≤ 0.05; ns > 0.05. Error bars represent standard error.

Figure 4

Characterization of FAO and respiratory chain complexes. (a) mRNA expression of various mitochondrial proteins, from different sections of the mitochondria, was analyzed using qPCR in patient and control fibroblasts. qPCR revealed reduced mRNA expression of all proteins tested compared to control. (b) Mitochondrial proteins involved in various pathways of mitochondrial respiration including FAO and respiratory chain were analyzed via SDS-PAGE western blotting with antigens for the corresponding proteins. Mitochondrial and cytosolic loading controls were used: anti-AK2 and anti-GAPDH antigens, respectively. Twenty-five μg protein was loaded for the anti-VLCAD, anti-MCAD, anti-ETFDH, anti-IVD, and anti-Hsp60 blots. Twenty μg protein was loaded for the respiratory chain complexes. Cropped images shown, full-length blots are presented in Supplementary Figs. S7–S9. (c) Immunofluorescent staining of fibroblasts from patients and a control using antigens for mitochondrial proteins VLCAD, MCAD, IVD, and ETFDH. These proteins were visualized with green fluorescently tagged antibodies to evaluate the presence of these proteins and nuclei were visualized with Hoescht-Blue stain. Images were taken at 60X magnification. p value: ****≤ 0.0001; ***≤ 0.001; **≤ 0.01; *≤ 0.05; ns > 0.05. Error bars represent standard error.