Impaired NADH-linked mitochondrial respiration disrupts ventral midbrain neuronal programs in POLG disease

POLG (DNA polymerase γ catalytic subunit)-related mitochondrial diseases are among the most severe primary mitochondrial disorders and are characterized by progressive neurodegeneration with prominent dopaminergic involvement. However, the cell type-specific mechanisms linking mitochondrial DNA instability to neuronal vulnerability remain incompletely defined.

Using patient-derived midbrain organoids and single-cell RNA sequencing, we investigated how POLG mutations alter mitochondrial and neuronal programs at subtype resolution. We analyzed dopaminergic neuronal populations and ventral midbrain neurons to define disease-associated transcriptional changes. To evaluate therapeutic improvement, POLG organoids were treated chronically with nicotinamide riboside (NR), followed by single-cell transcriptomic profiling and pathway enrichment analysis.

POLG mutations induced a coordinated downregulation of genes associated with oxidative phosphorylation and synaptic signaling, particularly in terminally differentiated dopaminergic neurons. This transcriptional alteration involved genes encoding respiratory chain complexes I-V, mitochondrial translation machinery, and ATP synthase components, suggesting disruption of mitochondrial bioenergetic programs at the transcriptomic level. Among dopaminergic subtypes, DA2 neurons and ventral midbrain neurons showed the most pronounced transcriptional alterations, indicating maturation-dependent vulnerability. NR treatment was associated with altered expression of genes involved in oxidative phosphorylation, NADH dehydrogenase activity, respiratory chain assembly, and synaptic pathways. Following NR exposure, dopaminergic subpopulations exhibited changes in cell-type proportions and partial normalization of mitochondrial- and synaptic-related transcriptional programs.

These findings identify transcriptional alterations in pathways related to mitochondrial respiration. The data further suggests that modulation of NAD⁺ metabolism is associated with transcriptional changes in mitochondrial and neuronal pathways in this disease context.