Metformin boosts mitochondria and neurogenesis via AMPK/mTOR/SIRT3 in POLG mutant organoids

Mutations in the POLG gene, encoding the catalytic subunit of mitochondrial DNA polymerase gamma, are the most common cause of mitochondrial diseases affecting the central nervous system. These mutations frequently result in neurodevelopmental disorders, yet the cellular and molecular mechanisms underlying POLG related encephalopathies remain poorly understood. In particular, how POLG mutations affect mitochondrial function, neural progenitor behavior, and early neurogenesis in the developing human brain has not been fully elucidated.

Methods

To investigate the impact of POLG mutations on human neurodevelopment, we generated 3D cortical brain organoids from induced pluripotent stem cells (iPSCs) derived from a patient carrying compound heterozygous POLG mutations (A467T/W748S). Organoid development was monitored using immunohistochemistry, transmission electron microscopy, and live-cell mitochondrial assays. Single-cell RNA sequencing (scRNA-seq) was performed to profile cellular diversity and transcriptional changes. Organoids were treated with metformin, a known mitochondrial modulator, and mitochondrial function was assessed by measuring membrane potential (TMRE), ATP production, and mtDNA copy number. Western blotting and immunofluorescence were used to investigate AMPK–SIRT3–mTOR signaling and markers of mitochondrial dynamics and mitophagy. Statistical analyses were performed using unpaired t-tests or ANOVA, with p-values < 0.05 considered significant.

Results

POLG mutant cortical organoids exhibited impaired neural differentiation, with expansion of stress-associated neural progenitors and reduced neuronal populations. scRNA seq analysis revealed transcriptional signatures of oxidative stress, mitochondrial dysfunction, and suppressed neurogenesis, particularly in a specific neural progenitor subpopulation. Metformin treatment significantly improved mitochondrial membrane potential, ATP output, and mtDNA copy number in POLG organoids. It also promoted neuronal differentiation and reduced reactive progenitor states. Mechanistically, metformin activated AMPK and SIRT3, inhibited mTOR, enhanced expression of mitochondrial fusion and biogenesis markers (OPA1, PGC-1α), and increased autophagic and mitophagic activity (LC3B, BNIP3).

Conclusions

Our study demonstrates that POLG mutations disrupt early cortical development by impairing mitochondrial function and skewing progenitor fate. Metformin mitigates these effects by restoring mitochondrial homeostasis and promoting neurogenesis through the AMPK/SIRT3/mTOR axis. These findings offer mechanistic insights into POLG-related encephalopathy and support metformin as a candidate for therapeutic intervention in mitochondrial neurodevelopmental disorders.