Small molecules rescue pathogenic POLGA aggregation and restore its functions in a mouse model of PEO

Abstract POLGA, the catalytic subunit of the mitochondrial DNA polymerase, is essential for mitochondrial DNA (mtDNA) replication and maintenance. Mutations in POLGA cause progressive external ophthalmoplegia (PEO), but the underlying pathogenic mechanisms remain unclear. Here, we show that the mitochondrial E3 ligase MITOL hyperubiquitylates PEO-associated POLGA mutants at lysine 1060, triggering conformational changes and formation of insoluble aggregates that impair their mitochondrial import and consequently compromise mtDNA replication and mitochondrial homeostasis. To identify compounds that disrupt POLGA aggregates and restore mutant POLGA function, we screened an FDA/EMA/PMDA-approved drug library using a split-GFP system. We identified two clinically approved compounds, diltiazem hydrochloride (C1) and sulbactam (C4), that disrupted POLGA aggregates, prevented aberrant MITOL recognition, restored mitochondrial import, and rescued mtDNA replication and mitochondrial function. Importantly, treatment with either compound significantly improved motor performance in a knock-in mouse model carrying the disease-relevant A467T-equivalent POLGA mutation. These findings establish pathogenic aggregation as a previously underrecognized disease mechanism and demonstrate that disrupting POLGA aggregates with repurposed drugs can restore mitochondrial function in vivo, providing a potential therapeutic strategy for POLGA-associated mitochondrial disease.