Modelling POLG mutations in mice: pathogenesis and mechanisms

Mitochondrial disorders (MDs) are a heterogeneous group of metabolic diseases caused by pathogenic variants in mitochondrial DNA (mtDNA) or in nuclear genes encoding proteins essential for mitochondrial function. Among these, mutations in the POLG gene, encoding the catalytic subunit of DNA polymerase γ, represent the most common cause of inherited mitochondrial disease related to a single nuclear gene. This thesis aims to investigate the molecular mechanisms and pathological consequences of POLG mutations by generating and characterizing novel mouse models and performing complementary in vitro analyses. This work focused on the three most common recessive mutations in POLG (A467T, W748S, G848S) as well as the major dominant one (Y955C). In particular, I characterized (i) homozygous knock-in mutants for the single mutations, (ii) compound heterozygous mice carrying one mutant allele and one null knock-out (KO) allele, and (iii) compound heterozygous mutants combining different pathogenic variants. Consistent with the severity reported in patients, four out of nine allelic combinations proved lethal during the embryonic development. The remaining five murine models were viable and provided a valuable platform to study POLG disorders. I carried out molecular analyses to assess mtDNA content and integrity in high-energy-demanding tissues, along with measurements of respiratory chain complex activities in tissues showing mtDNA depletion. Since the Y955C mutation leads to late-onset phenotypes in humans, Polg+/Y933C mice were also characterized at 24 months of age. Heterozygous animals performed normally across various in vivo tests and showed normal mtDNA content and integrity. By contrast, histological and ultrastructural analyses revealed a late-onset phenotype affecting brain, liver and skeletal muscle. In particular, H&E staining showed increased vacuolization in the white matter of the cerebellum and the brainstem, accompanied by increased immunohistochemical staining for CD68 and GFAP in the same regions. In skeletal muscle, COX/SDH histochemistry highlighted the presence of fibers with mitochondria-depleted areas in the Polg+/Y933C mutants, while transmission electron microscopy (TEM) revealed a higher proportion of fibers with aberrant mitochondria and altered ultrastructure. Complementary in vitro analyses and structural studies elucidated how the different mutations impacted polymerase activity, unravelling species-specific differences between the human and murine enzymes. These analyses provided a mechanistic explanation for the stronger stimulatory effect of the murine POLγB accessory subunit, which may account for the milder phenotypes observed in mice. In conclusion, this work provides novel insights into the pathogenesis of POLG-related disorders, thereby establishing robust murine models for preclinical studies and highlighting potential targets for therapeutic intervention.