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Updated: Jul 5, 2026
An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model
Published on: March 9, 2022
MELAS mitochondrial DNA mutation A3243G reduces glutamate transport in cybrids cell lines
Jacopo C DiFrancesco1, J Mark Cooper, Amanda Lam
1Department of Neuroscience and Biomedical Technologies, University of Milano-Bicocca, Monza, Italy. jacopo.difrancesco@unimib.it
Abstract:
MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) is commonly associated with the A3243G mitochondrial DNA (mtDNA) mutation encoding the transfer RNA of leucine (UUR) (tRNA (Leu(UUR))). The pathogenetic mechanisms of this mutation are not completely understood. Neuronal functions are particularly vulnerable to alterations in oxidative phosphorylation, which may affect the function of the neurotransmitter glutamate, leading to excitotoxicity. In order to investigate the possible effects of A3243G upon glutamate homeostasis, we assessed glutamate uptake in osteosarcoma-derived cytoplasmic hybrids (cybrids) expressing high levels of this mutation. High-affinity Na(+)-dependent glutamate uptake was assessed as radioactive [(3)H]-glutamate influx mediated by specific excitatory amino acid transporters (EAATs). The maximal rate (V(max)) of Na(+)-dependent glutamate uptake was significantly reduced in all the mutant clones. Although the defect did not relate to either the mutant load or magnitude of oxidative phosphorylation defect, we found an inverse relationship between A3243G mutation load and mitochondrial ATP synthesis, without any evidence of increased cellular or mitochondrial free radical production in these A3243G clones. These data suggest that a defect of glutamate transport in MELAS neurons may be due to decreased energy production and might be involved in mediating the pathogenic effects of the A3243G mtDNA mutation.
Insights
The A3243G mitochondrial DNA mutation in MELAS may impair neuronal glutamate uptake due to reduced energy production, not free radical damage. This defect could contribute to MELAS pathogenesis.
Area of Science:
- Neuroscience
- Mitochondrial Biology
- Genetics
Background:
- MELAS syndrome is linked to the A3243G mtDNA mutation affecting tRNA (Leu(UUR)).
- Neuronal excitotoxicity from glutamate dysregulation is a potential pathogenic mechanism.
- The precise impact of the A3243G mutation on glutamate homeostasis remains unclear.
Purpose of the Study:
- To investigate the effect of the A3243G mutation on glutamate uptake in neuronal models.
- To explore the relationship between glutamate transport defects, energy production, and oxidative stress in MELAS.
Main Methods:
- Utilized osteosarcoma-derived cybrid cells expressing the A3243G mtDNA mutation.
- Assessed high-affinity Na(+)-dependent glutamate uptake via [(3)H]-glutamate influx.
- Measured V(max) of glutamate uptake, mutant load, oxidative phosphorylation, ATP synthesis, and free radical production.
Main Results:
- Significantly reduced maximal rate (V(max)) of Na(+)-dependent glutamate uptake in all mutant cybrid clones.
- Found an inverse relationship between A3243G mutation load and mitochondrial ATP synthesis.
- Observed no increase in cellular or mitochondrial free radical production in A3243G clones.
Conclusions:
- Defective glutamate transport in MELAS neurons may stem from impaired mitochondrial energy production.
- This glutamate transport defect is a potential contributor to the pathogenicity of the A3243G mtDNA mutation.
- Findings suggest energy deficit, rather than oxidative stress, is a key factor in A3243G-related neuronal dysfunction.
