An anaplerotic approach to correct the mitochondrial dysfunction in ataxia-telangiectasia (A-T)

Abstract

Background: ATM, the protein defective in the human genetic disorder, ataxia-telangiectasia (A-T) plays a central role in response to DNA double-strand breaks (DSBs) and in protecting the cell against oxidative stress. We showed that A-T cells are hypersensitive to metabolic stress which can be accounted for by a failure to exhibit efficient endoplasmic reticulum (ER)-mitochondrial signalling and Ca2⁺ transfer in response to nutrient deprivation resulting in mitochondrial dysfunction. The objective of the current study is to use an anaplerotic approach using the fatty acid, heptanoate (C7), a metabolic product of the triglyceride, triheptanoin to correct the defect in ER-mitochondrial signalling and enhance cell survival of A-T cells in response to metabolic stress.

Methods: We treated control cells and A-T cells with the anaplerotic agent, heptanoate to determine their sensitivity to metabolic stress induced by inhibition of glycolysis with 2- deoxyglucose (2DG) using live-cell imaging to monitor cell survival for 72 h using the Incucyte system. We examined ER-mitochondrial signalling in A-T cells exposed to metabolic stress using a suite of techniques including immunofluorescence staining of Grp75, ER-mitochondrial Ca²⁺ channel, the VAPB-PTPIP51 ER-mitochondrial tether complexes as well as proximity ligation assays between Grp75-IP3R1 and VAPB1-PTPIP51 to establish a functional interaction between ER and mitochondria. Finally, we also performed metabolomic analysis using LC-MS/MS assay to determine altered levels of TCA intermediates A-T cells compared to healthy control cells.

Results: We demonstrate that heptanoate corrects all aspects of the defective ER-mitochondrial signalling observed in A-T cells. Heptanoate enhances ER-mitochondrial contacts; increases the flow of calcium from the ER to the mitochondrion; restores normal mitochondrial function and mitophagy and increases the resistance of ATM-deficient cells and cells from A-T patients to metabolic stress-induced killing. The defect in mitochondrial function in ATM-deficient cells was accompanied by more reliance on aerobic glycolysis as shown by increased lactate dehydrogenase A (LDHA), accumulation of lactate, and reduced levels of both acetyl CoA and ATP which are all restored by heptanoate.

Conclusions: We conclude that heptanoate corrects metabolic stress in A-T cells by restoring ER-mitochondria signalling and mitochondrial function and suggest that the parent compound, triheptanoin, has immense potential as a novel therapeutic agent for patients with A-T.

Keywords: ATM; Ataxia-telangiectasia; Endoplasmic reticulum–mitochondrial interaction; Heptanoate (C7); Mitochondrial dysfunction; Nutrient deprivation.

Figure 1

Correction of hypersensitivity to nutrient stress (glycolysis inhibition) and elevated ROS in A-T cells by heptanoate (C7). (A) Correction of sensitivity to nutrient stress in ATM-deficient cells by C7. HBEC (control), B3 (ATM-deficient cells generated by CRISPR/Cas9). 2DG, 2 deoxy glucose; C7, heptanoate. (B) Correction of sensitivity to nutrient stress by C7 in primary airway epithelial cells. Mean of two controls (C215, C218) and two A-T patient cells (AT009, AT014). (C) Correction of sensitivity in glucose-deprived olfactory neurosphere (ONS)-derived cells by C7. (D) Determination of mitochondrial ROS using MitoSOX labelling. NAC, N-acetylcysteine anti-oxidant. (E) Quantitation of the MitoSOX data was carried out by measuring fluorescence intensity for at least 4 separate determinations of cells in defined areas. (F) Correction of cell survival in control and ATM-deficient cells in response to NAC co-treatment with 2DG. C7 is included as a positive control. Scale bar, 5 μm. All data are plotted as mean +/− SEM. n ≥ 3, ∗p < 0.05 and ns for p > 0.05 using unpaired two-tailed Student's t-test (E) and two-way ANOVA (A,B,C and F).

Figure 2

Correction of ER-mitochondrial contact sites in ATM-deficient cells by C7. (A) Electron microscopy of HBEC control and ATM-deficient B3 cells with and without exposure to 2DG +/− C7. A distance of 25 nm between mitochondria and ER is considered a contact. Each contact site is highlighted with a red arrow. An increase in the number of contact sites was observed in HBEC cells exposed to 2DG but not in B3, whereas the opposite was observed for B3. However, treatment with C7 significantly increased those in B3 to control levels, n = 3 experiments, 2DG and C7 treatments were done for 4 h. (C) Co-staining using an antibody against the ER protein VAPB (green) and the mitochondrial protein PTPIP51 (red) was performed on HBEC and B3 cells in the presence or absence of 2DG. An increase in the fluorescence intensity was observed in HBEC cells following exposure to 2DG and not in B3 but this was corrected by C7. (D) Quantitation of fluorescence intensity for VAPB (D) and PTPIP51 (E) were carried out on at least 10 separate determinations of cells in defined areas. (F) Proximity ligation assay (PLA) using antibodies against VAPB (ER) and PTPIP51 (mitochondria) was performed as a means of showing increased interaction between these two proteins in the presence of C7. An increase in the number of foci in HBEC cells following exposure to 2DG was observed but not in B3. In this case, also C7 enhanced the number of foci in 2DG -treated B3 cells. (G) Quantitation of the number of foci per cell. (H) Immunoblotting for PTPIP51 and VAPB in cells following exposure to 2DG and C7. p97 protein was used as a loading control. Scale bar, 5 μm. Negative control with no primary antibody was performed. All data are plotted as mean +/-SEM. n ≥ 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 and ns for p > 0.05 using unpaired two-tailed Student's t-test.

Figure 3

Effect of C7 on the IP3R1-GRP75-VDAC1 Ca²⁺channel between ER and mitochondria. (A) Co-staining using an antibody against Grp75 (green) and TOMM20 (red) was performed on HBEC and B3 cells in the presence or absence of 2DG and C7. An increase in the fluoresce intensity was observed in HBEC cells following exposure to 2DG and not in B3 but the signal was enhanced to control levels in these cells in response to C7. (B) Quantitation of fluorescence intensity for Grp75 shows that C7 significantly increased the intensity of GRP75 labelling in ATM-deficient cells treated with 2DG, Scale bar, 5 μm (C) Proximity ligation assay (PLA) was also carried out here to confirm increased interaction between GRP75 and IP3R1. Unlike that in HBEC cells, an increase in the number of foci in ATM-deficient cells was not observed after 2DG but in the presence of C7, the number of foci was comparable to 2 DG-treated controls. (D) Quantitation of PLA foci per cell. Scale bar, 5 μm. (E) Immunoblotting for Grp75, IP3R1, and VDAC1 in cells following exposure to 2DG and C7 treatment again confirmed ATM activation after nutrient stress but showed no differences in IP3R1-GRP75-VDAC1 protein levels after 2DG treatment or when C7 was added. p97 protein was used as a loading control. All data are plotted as mean +/− SEM. n ≥ 3, ∗p < 0.05, ns for p > 0.5 using unpaired two-tailed Student's t-test.

Figure 4

Correction of Ca²⁺transfer between ER and mitochondria in ATM-deficient cells by C7. (A) Determination of intracellular calcium using FURA-2, a sensitive indicator for Ca²⁺ largely released from intracellular ER stores. While the release was deficient in ATM-deficient cells after 2DG treatment this was restored to control levels in the presence of C7. (B) In order to determine Ca²⁺ uptake into mitochondria, we employed Rhodamine-2AM a cationic fluorescent dye that labels respiring mitochondria. Similar results to those with FURA-2 were observed., a significant increase in calcium uptake occurred in ATM-deficient treated with 2DG in the presence of C7 indicating that C7 corrected both release and uptake of Ca²⁺. All data are plotted as mean +/− SEM. n ≥ 3, ∗∗∗∗p < 0.0001, ns for p > 0.5 using two-way ANOVA.

Figure 5

Correction of mitochondrial dysfunction in ATM-deficient cells by C7. (A) Mitochondrial function analysis was performed using the Seahorse XF24 extracellular flux analyser. 2DG causes a major increase in oxygen consumption rate (OCR) and respiratory capacity in ATM-deficient cells which was reduced to normal levels with C7 co-treatment. (C) Significant reduction in basal OCR by C7 in untreated ATM-deficient cells. (D) Determination of ATP levels at 1 h after exposure to 2DG in the presence and absence of C7. (E) Determination of ATP levels at 5 h after exposure to 2DG in the presence and absence of C7. ATP was assayed using a colorimetric method as described in Methods. All data are plotted as mean +/− SD, n ≥ 3, ∗p < 0.01, ns for p > 0.05 using two-way ANOVA (A, B and C) and unpaired two-tailed Student's t-test (D, E, and F).

Figure 6

Metabolic analysis of glycolysis intermediates in 2DG and 2 DG + C7 treated cells. (A) Quantification of LDHA1 mRNA fold changes in HBEC and B3 cells was carried out by qPCR. Fold changes are shown as 2-ɗɗCt normalised to housekeeping gene GAPDH. LDHA1 mRNA levels were ∼20 fold higher in B3 cells which further elevated to ∼35-fold with 2 DG treatment. Exposure to C7 treatment brought the LDHA1 expression down to levels comparable to HBEC (∼10-fold). (B) Quantitation of lactate levels in HBEC and B3 cells was carried out by LC-MS analysis. A significant increase in lactate levels was observed in untreated B3 cells compared to HBEC cells. Lactate levels were further enhanced in B3 cells treated with 2DG while the levels remained unaltered in HBEC cells. Exposure to C7 treatment brought down the lactate concentration in B3 cells to levels comparable to HBEC cells which were also similar to levels observed in untreated HBEC cells. (C) Quantitation of pyruvate levels in HBEC and B3 cells. Pyruvate levels were significantly lower in untreated B3 cells compared to HBEC controls. Pyruvate levels increased significantly in response to 2DG treatment in HBEC cells whereas the levels remained unaltered in B3 cells. Exposure to C7 treatment caused a significant increase of pyruvate concentration in B3 cells to levels comparable to HBEC controls. (D) Quantitation of acetyl CoA levels showed a significant decrease in B3 cells compared to HBEC cells. Exposure to C7 treatment caused a significant increase in acetyl CoA concentration in B3 cells to levels comparable to HBEC controls. (E) Quantitation of ATP showed a significant decrease in B3 cells compared to HBEC cells. Exposure to C7 treatment caused a significant increase in ATP concentration in B3 cells to levels comparable to HBEC controls. All data are plotted as mean +/− SEM. n ≥ 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 unpaired two-tailed Student's t-test.

Figure 7

Anaplerotic effect of heptanoate in correcting the mitochondrial defect in ataxia-telangiectasia (A–T) cells. Inhibition of glycolysis with 2DG treatment and the resulting nutrient stress activates ATM and enhances ER-mitochondrial signalling for the transfer of Ca²⁺ between the two organelles. This signalling is deficient in A-T cells rendering them hypersensitive to this form of metabolic stress. Co-treatment of ATM-deficient cells with heptanoate (C7, a product of triheptanoin metabolism) corrects all aspects of the signalling defect and restores sensitivity to metabolic stress to control levels. The underlying mechanism is that heptanoate is converted to acetyl CoA and propionyl CoA, both of which enter and help boost the TCA cycle and correct the mitochondrial function in ATM-deficient cells. More functional mitochondria lead to enhanced interaction with the ER which increases the transfer of Ca²⁺ to promote cell survival. These results suggest that heptanoate by increasing access to TCA intermediates improves mitochondrial efficiency and in turn improves ER-mitochondrial interaction and cell bioenergetics. The TCA cycle depicted here is a simpler version involving some of the key metabolites and is not comprehensive.