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Updated: Sep 2, 2025
An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model
Published on: March 9, 2022
Rapamycin rescues mitochondrial dysfunction in cells carrying the m.8344A > G mutation in the mitochondrial tRNALys
Mariantonietta Capristo1, Valentina Del Dotto1, Concetta Valentina Tropeano1
1IRCCS Istituto delle Scienze Neurologiche di Bologna, Programma di Neurogenetica, via Altura 3, 40139, Bologna, Italy.
Background:
Myoclonus, Epilepsy and Ragged-Red-Fibers (MERRF) is a mitochondrial encephalomyopathy due to heteroplasmic mutations in mitochondrial DNA (mtDNA) most frequently affecting the tRNALys gene at position m.8344A > G. Defective tRNALys severely impairs mitochondrial protein synthesis and respiratory chain when a high percentage of mutant heteroplasmy crosses the threshold for full-blown clinical phenotype. Therapy is currently limited to symptomatic management of myoclonic epilepsy, and supportive measures to counteract muscle weakness with co-factors/supplements.
Methods:
We tested two therapeutic strategies to rescue mitochondrial function in cybrids and fibroblasts carrying different loads of the m.8344A > G mutation. The first strategy was aimed at inducing mitochondrial biogenesis directly, over-expressing the master regulator PGC-1α, or indirectly, through the treatment with nicotinic acid, a NAD+ precursor. The second was aimed at stimulating the removal of damaged mitochondria through prolonged rapamycin treatment.
Results:
The first approach slightly increased mitochondrial protein expression and respiration in the wild type and intermediate-mutation load cells, but was ineffective in high-mutation load cell lines. This suggests that induction of mitochondrial biogenesis may not be sufficient to rescue mitochondrial dysfunction in MERRF cells with high-mutation load. The second approach, when administered chronically (4 weeks), induced a slight increase of mitochondrial respiration in fibroblasts with high-mutation load, and a significant improvement in fibroblasts with intermediate-mutation load, rescuing completely the bioenergetics defect. This effect was mediated by increased mitochondrial biogenesis, possibly related to the rapamycin-induced inhibition of the Mechanistic Target of Rapamycin Complex 1 (mTORC1) and the consequent activation of the Transcription Factor EB (TFEB).
Conclusions:
Overall, our results point to rapamycin-based therapy as a promising therapeutic option for MERRF.
Insights
Rapamycin treatment shows promise for Myoclonus, Epilepsy and Ragged-Red-Fibers (MERRF) by improving mitochondrial function. This therapy enhanced respiration in cells with MERRF mutations, offering a potential new treatment option.
Area of Science:
- Mitochondrial Medicine
- Genetics
- Cell Biology
Background:
- Myoclonus, Epilepsy and Ragged-Red-Fibers (MERRF) is a mitochondrial disorder caused by mutations in mitochondrial DNA (mtDNA), primarily affecting the tRNA^Lys gene.
- The m.8344A>G mutation impairs mitochondrial protein synthesis and respiratory function, leading to severe clinical symptoms when heteroplasmy levels are high.
- Current treatments for MERRF are limited to symptom management and supportive care.
Purpose of the Study:
- To evaluate therapeutic strategies for rescuing mitochondrial function in MERRF.
- To investigate the efficacy of inducing mitochondrial biogenesis and promoting mitophagy in cellular models of MERRF.
Main Methods:
- Tested two strategies: 1) inducing mitochondrial biogenesis (via PGC-1α or nicotinic acid) and 2) stimulating mitophagy (via prolonged rapamycin treatment).
- Assessed mitochondrial function in cybrids and fibroblasts with varying loads of the m.8344A>G mutation.
Main Results:
- Mitochondrial biogenesis induction showed limited efficacy, especially in high-mutation load cells.
- Chronic rapamycin treatment significantly improved mitochondrial respiration in fibroblasts with intermediate and high mutation loads, completely rescuing bioenergetic defects in the latter.
- Rapamycin's effects were linked to increased mitochondrial biogenesis, potentially via mTORC1 inhibition and TFEB activation.
Conclusions:
- Rapamycin-based therapy represents a promising therapeutic avenue for MERRF.
- Targeting mitophagy may be a more effective strategy than solely inducing mitochondrial biogenesis for MERRF treatment.
