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Updated: Jun 5, 2025
Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima
Published on: August 12, 2018
Inhibition of the PI3K-AKT-MTORC1 axis reduces the burden of the m.3243A>G mtDNA mutation by promoting mitophagy and
Chih-Yao Chung1, Kritarth Singh1, Preethi Sheshadri1
1Department of Cell and Developmental Biology and Consortium for Mitochondrial Research, UCL, London, UK.
Abstract:
Mitochondrial DNA (mtDNA) encodes genes essential for oxidative phosphorylation. The m.3243A>G mutation causes severe disease, including myopathy, lactic acidosis and stroke-like episodes (MELAS) and is the most common pathogenic mtDNA mutation in humans. We have previously shown that the mutation is associated with constitutive activation of the PI3K-AKT-MTORC1 axis. Inhibition of this pathway in patient fibroblasts reduced the mutant load, rescued mitochondrial bioenergetic function and reduced glucose dependence. We have now investigated the mechanisms that select against the mutant mtDNA under these conditions. Basal macroautophagy/autophagy and lysosomal degradation of mitochondria were suppressed in the mutant cells. Pharmacological inhibition of any step of the PI3K-AKT-MTORC1 pathway activated mitophagy and progressively reduced m.3243A>G mutant load over weeks. Inhibition of autophagy with bafilomycin A1 or chloroquine prevented the reduction in mutant load, suggesting that mitophagy was necessary to remove the mutant mtDNA. Inhibition of the pathway was associated with metabolic remodeling - mitochondrial membrane potential and respiratory rate improved even before a measurable fall in mutant load and proved crucial for mitophagy. Thus, maladaptive activation of the PI3K-AKT-MTORC1 axis and impaired autophagy play a major role in shaping the presentation and progression of disease caused by the m.3243A>G mutation. Our findings highlight a potential therapeutic target for this otherwise intractable disease.Abbreviation: ΔΨm: mitochondrial membrane potential; 2DG: 2-deoxy-D-glucose; ANOVA: analysis of variance; ARMS-qPCR: amplification-refractory mutation system quantitative polymerase chain reaction; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CQ: chloroquine; Cybrid: cytoplasmic hybrid; CYCS: cytochrome c, somatic; DCA: dichloroacetic acid; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethylsulfoxide; EGFP: enhanced green fluorescent protein; LC3B-I: carboxy terminus cleaved microtubule-associated protein 1 light chain 3 beta; LC3B-II: lipidated microtubule-associated protein 1 light chain 3 beta; LY: LY290042; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MELAS: mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes; MFC: mitochondrial fragmentation count; mt-Keima: mitochondrial-targeted mKeima; mtDNA: mitochondrial DNA/mitochondrial genome; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OA: oligomycin+antimycin A; OxPhos: oxidative phosphorylation; DPBS: Dulbecco's phosphate-buffered saline; PPARGC1A/PGC-1α: PPARG coactivator 1 alpha; PPARGC1B/PGC-1β: PPARG coactivator 1 beta; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; qPCR: quantitative polymerase chain reaction; RNA-seq: RNA sequencing; RP: rapamycin; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; WT: wild-type.
Insights
Inhibiting the PI3K-AKT-MTORC1 pathway in cells with the m.3243A>G mitochondrial DNA mutation activates mitophagy. This process clears mutant mtDNA, improving mitochondrial function and offering a potential therapeutic strategy for MELAS and related diseases.
Area of Science:
- Cellular Biology
- Mitochondrial Genetics
- Molecular Medicine
Background:
- The m.3243A>G mutation in mitochondrial DNA (mtDNA) is a leading cause of severe human diseases like MELAS.
- This mutation is linked to overactivation of the PI3K-AKT-MTORC1 signaling pathway.
- Previous studies showed inhibiting this pathway in patient cells reduced mutant mtDNA load and improved mitochondrial function.
Purpose of the Study:
- To investigate the mechanisms by which inhibiting the PI3K-AKT-MTORC1 pathway reduces mutant mtDNA load.
- To determine the role of autophagy, specifically mitophagy, in the clearance of m.3243A>G mutant mtDNA.
- To understand the metabolic changes associated with pathway inhibition and their contribution to disease amelioration.
Main Methods:
- Utilized patient-derived fibroblasts carrying the m.3243A>G mtDNA mutation.
- Pharmacologically inhibited the PI3K-AKT-MTORC1 pathway using various inhibitors.
- Assessed mitophagy using specific inhibitors (bafilomycin A1, chloroquine) and measured mutant mtDNA load via qPCR.
- Evaluated mitochondrial function, including membrane potential and respiratory rate, and metabolic remodeling.
Main Results:
- Inhibition of the PI3K-AKT-MTORC1 pathway activated mitophagy, leading to a progressive reduction in m.3243A>G mutant mtDNA over weeks.
- Blocking autophagy prevented the clearance of mutant mtDNA, confirming mitophagy's essential role.
- Metabolic improvements, such as enhanced mitochondrial membrane potential and respiratory rate, occurred even before significant mutant load reduction and were crucial for mitophagy.
- Impaired autophagy was identified as a key factor in the disease presentation and progression.
Conclusions:
- Maladaptive activation of the PI3K-AKT-MTORC1 pathway and suppressed mitophagy contribute significantly to the pathology of m.3243A>G mtDNA diseases.
- Targeting the PI3K-AKT-MTORC1 pathway and enhancing mitophagy presents a promising therapeutic strategy for intractable mitochondrial disorders.
- Metabolic remodeling plays a critical role in facilitating mitophagy and rescuing mitochondrial function.
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