Myoclonic epilepsy with ragged-red fibers: advancing personalized medicine through iPSC manipulation and neural modelling

Myoclonic Epilepsy with Ragged-Red Fibers (MERRF) is a rare genetic condition mostly associated with the m.8344A>G substitution within the mitochondrial MT-TK gene, which codes for mt-tRNALys. The biochemical dysfunctions encompass premature mitochondrial protein termination, aberrant OXPHOS subunit polypeptides and bioenergetic failure, ensuing in increased oxidative stress, inflammation and progressive neurodegeneration. Nevertheless, the overarching pathogenic mechanisms of the disease remain elusive. This study aimed to generate MERRF induced pluripotent stem cells (iPSCs) from patient-derived somatic cells, expanding on the fine-tuning of alternative approaches to shift heteroplasmy artificially. MERRF iPSCs were leveraged for neural differentiation into neural progenitors and cortical organoids, as well as for the repurposing of rapamycin as a therapeutic strategy. However, the reprogramming efficiency to high-heteroplasmy iPSCs was negligible. Low-heteroplasmy cortical organoids were generated, but only revealed a decline in the expression of common NPC and synaptic markers, as well as morphological aberrations associated with an astrocytic depletion. Inducing a genetic bottleneck in iPSCs with ethidium bromide allowed to obtain multiple clones with variable m.8344A>G load proportions. Conversely, mitochondrial transfer from high-heteroplasmy cybrid donor cells to isogenic wild-type or intermediate-heteroplasmy iPSC recipients proved inefficient. NPCs generated from intermediate and high-heteroplasmy MERRF iPSCs showed an increased mitochondrial DNA content, while oxygen consumption revealed an increment in the steady-state levels of basal respiration in high-heteroplasmy NPCs. Rapamycin treatment led to an increase in mtDNA copy number associated with improved respiration only for the intermediate MERRF NPC cell line. MERRF cortical organoids were established from high-heteroplasmy iPSCs to optimize rapamycin treatment, which indicated that late-stage incubation is recommended to avoid developmental inhibition. In conclusion, this study presented an efficient method to circumvent the challenges associated with high-heteroplasmy iPSC reprogramming and introduced 3D cortical organoids for MERRF, which will be used for further investigations into the potential therapeutic effects of rapamycin.