Exploring the Interplay between Mitochondrial Heteroplasmy and Cell Signaling Pathways

Chung, Chih-Yao;


Exploring the Interplay between Mitochondrial Heteroplasmy and Cell Signaling Pathways.

Doctoral thesis (Ph.D), UCL (University College London).


– Accepted Version

Access restricted to UCL open access staff until 1 June 2023.

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Introduction: Mutations of the mitochondrial genome (mtDNA) cause many profoundly debilitating clinical conditions with limited treatment options. Most mtDNA diseases show heteroplasmy – tissues express both wild-type and mutant mtDNA. While the level of heteroplasmy broadly correlates with disease severity, the relationships between specific mtDNA mutations, heteroplasmy, disease phenotype and severity are not well-understood. Among human pathogenic mtDNA mutations, the m.3243A>G mutation is the most prevalent and the primary cause of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS).
Aims: I aimed to carry out extensive bioenergetic, metabolomic and transcriptomic studies on heteroplasmic patient-derived fibroblasts and A549 cybrid cells carrying the m.3243A>G mutation and to establish causality/links among mtDNA heteroplasmy, metabolic phenotypes and alternations in cell signalling.
Results: Using live-cell imaging, I found that the m.3243A>G mutation increases glucose dependence in the mutant cells, resulting in redox imbalance and oxidative stress. Metabolomics further revealed that the m.3243A>G mutation remodels glucose and lipid metabolism towards increased anabolic biosynthesis and lipid accumulation. Moreover, RNA-sequencing and immunofluorescence showed that the m.3243A>G mutation leads to changes in cell signalling, promoting the upregulation of the PI3K-Akt-mTORC1 axis in patient-derived cells and tissues. Remarkably, pharmacological inhibition of PI3K, Akt, or mTORC1 for 6-12 weeks activated mitophagy, reduced mtDNA mutant load and rescued cellular bioenergetics. I further established that the reduction of the mutant load is cell-autonomous by long-term cell growth/death analysis and single-cell PCR. In addition, the decline was prevented by inhibition of mitophagy, showing that mitophagy is necessary. Of note, I also examined the effects of the PI3K-Akt-mTORC1 inhibition in cells carrying the m.8993T>G mtDNA point mutation. In these cells, inhibition of the axis had no impact on mutant load. The result suggests that hyperactivation of the PI3K-Akt-mTORC1 axis is relatively disease-specific and perhaps points towards the mechanisms that define different disease phenotypes between various mitochondrial diseases, emphasising that therapeutic options should be considered separately for each disease-related to mtDNA mutations.
Conclusion: Together, these data strongly argue that the chronic activation of the PI3K-Akt-mTORC1 axis, presumably as an adaptive response to impaired oxidative metabolism, instead serves as a maladaptive response in the m.3243A>G mutation disease model and thus represents a therapeutic target with translational potential that may benefit people suffering from the consequences of the m.3243A>G mutation.

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