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 Ca 2+ 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.

In Cerebellar Atrophy of 12-Month-Old ATM-Null Mice, Transcriptome Upregulations Concern Most Neurotransmission and Neuropeptide Pathways, While Downregulations Affect Prominently Itpr1, Usp2 and Non-Coding RNA

“…In the disease context, the transcriptome showed a dysregulated expression with nominal significance for genes responsible for phenotypes of ataxia (compiled according to the Online Mendelian Inheritance of Man database, https://www.ncbi.nlm.nih.gov/omim/, accessed on 29 February 2022). Downregulations were observed for Atm, Itpr1, Syne1, Grid2, Grik2, Fgf14, Rora, Gba2, Reln, in good agreement with a previous proteome study of cerebrospinal fluid from A-T patients [67]; a significant enrichment was detected on the STRING webserver for "abnormal cerebellar granule neuron morphology" (q = 0.0014) for the cluster of ATM, RORA [74] and GRID2 [75] proteins; an enrichment for "postsynapse" (q = 0.0182) was detected for ITPR1 [66,67,75,76], SYNE1, GRID2 [75] and GRIK2 [77]; upregulations were observed for the ataxia genes Mme, Ebf3, Vamp1, Ppp2r2b, Svbp, without significant enrichment, but VAMP1 being a vesicle-associated factor like ATM. Significant expression changes existed also for genes responsible for the pathogenesis of telangiectasia (upregulation of Sst, Sstr1, Sstr2, Tac1, Tacr1, Svbp) [78][79][80][81], and for general growth (Sst, Sstr1, Sstr2) [82].…”

Metabolic Stress and Mitochondrial Dysfunction in Ataxia-Telangiectasia

“…Several parameters of mitochondrial dysfunction in ATM-deficient cells were also corrected by C7. Higher basal levels of lactate dehydrogenase (LDHA) and further increases in metabolic stress, together with increases in lactate, supported a greater reliance on aerobic glycolysis in ATM-deficient cells [17]. Compatible with the anaplerotic effects of C7, the inclusion of this compound dramatically reduced LDHA and lactate levels while improving mitochondrial function in A-T cells (Figure 6).…”

“…Since mitochondrial dysfunction and abnormalities in energy metabolism are frequently observed in A-T cells, they represent a potential target for an anaplerotic approach to correction. Yeo et al described the correction of ER-mitochondrial signaling and mitochondrial function in ATMdeficient cells using heptanoate [17]. They confirmed a hypersensitivity to nutrient deprivation in an A-T bronchial epithelial cell line, and that heptanoate (C7) reduced the extent Further, heptanoate that enters the cell is metabolized into acetyl-CoA, which acts as an anaplerotic fuel for the synthesis of citric acid.…”

“…In normal conditions, mitochondria produce reactive oxygen species (ROS), which participate in intracellular signaling [16]. On the other hand, a general feature of A-T cells is the elevated basal levels of ROS, which is thought to arise by mitochondrial dysfunction [17]. The observation that mitochondrial-derived H 2 O 2 produces ATM dimerization that is localized to the nucleus, suggests that ROS-mediated stress signaling relays mitochondria to the nucleus [18].…”

Ataxia Telangiectasia patient-derived neuronal and brain organoid models reveal mitochondrial dysfunction and oxidative stress

“…This reduction in the AT mutant organoids' capacity to respond to glutamate aligns with the GO term transcriptome analysis that identified the glutamate receptor signalling pathway as significantly downregulated in the mutant organoids (Fig 7G). Brain organoids were treated with the antioxidant NAC as well as the anaplerotic agent heptanoate (C7), which replenishes TCA cycle activity and was shown to alleviate mitochondrial dysfunction, oxidative stress and cell death in ATM -/-HBEC and AT patient-derived ONS cells under metabolic stress conditions [66]. Brain organoids treated with C7 and NAC did not show improvements in unstimulated neuronal firing rates (Fig 7I ), however when exposed to glutamate stimulation, the NAC and C7 treated AT mutant organoids had mean firing rates that were significantly increased compared to the untreated AT organoids, and in the case of NAC treatment, the firing rate was restored to that of the gene corrected organoids (Fig 7J ).…”