Mitochondrial DNA heteroplasmy drives cortical neuronal disturbances in human organoids harbouring the common m.3243A>G mutation

Mitochondrial diseases frequently affect the brain leading to severe and disabling neurological symptoms. The heteroplasmic m.3243A>G mutation in MT-TL1, encoding mt-tRNALeu, is responsible for ~80% of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), which is one of the most characteristic mitochondrial syndromes, leading to disability and early death. There are no animal models harbouring this mutation to provide precise mechanistic insights informing therapeutic interventions. Here, we generated a human iPSC-derived cerebral organoid slice model that recapitulates cortical architecture and mitochondrial pathology. Using biological assays and single-cell RNA sequencing, we uncovered heteroplasmy-dependent transcriptional shifts and changes in key cellular processes in cortical neurons. Organoids with high heteroplasmy showed a predominant impairment of deep-layer neurons triggered by mitochondrial stress, leading to axonal degeneration and apoptosis, similar to brain autopsy of a MELAS patient. Our findings provide insights into the vulnerability of long-range projection neurons in mitochondrial diseases, advancing our understanding of disease mechanisms with a view to potential therapeutic strategies.