Mouse models for the study of mitochondrial disorders

Leigh syndrome (LS), or subacute necrotizing encephalomyelopathy, is the most common mitochondrial disease in infancy. Both neurological signs and pathological lesions of Leigh disease are mimicked by the ablation of the mouse mitochondrial respiratory chain subunit NDUFS4, which is crucial for normal CI activity and assembly, particularly in the brain. Currently, no curative therapy for LS is available. In this thesis, I exploited a new, self-complementary adeno-associated viral 9 vector (scAAV9) to deliver the human NDUFS4 gene product in Ndufs4-/- mice. Either single intra-vascular (i.v.) or double i.v. and intra-cerebro-ventricular (i.c.v.) injections were performed at post-natal day 1 (P1). The first strategy doubled the lifespan of the Ndufs4-/- mice from 45 to ≈100 days after birth, when the mice developed rapidly progressive neurological failure. However, the double i.v. and i.c.v. administration of the scAAV9-hNDUFS4 prolonged healthy lifespan up to 9 months of age. These mice were well and active at euthanization performed to investigate the brain and other organs post-mortem. Robust expression of hNDUFS4 was detected in different cerebral areas preserving normal morphology and restoring Complex I activity and assembly. These data warrant further investigations to fully assess translatability of the scAAV9-hNDUFS4-based therapy to the patients by investigating its effectiveness during the prodromal phase of the disease in mice and eventually humans. Mutations in the POLG gene, coding for POLA, are a common cause of human disease leading to a spectrum of disorders characterized by mtDNA instability, that compromise the mitochondrial function. Despite being relatively frequent, the molecular pathogenesis of POLG-related diseases is poorly understood, and efficient treatments are missing, partly due to the lack of relevant in vivo models. In the second part of my dissertation, I discuss the in vivo and in vitro characterization of the POLA Y933C mutation, which reproduces the Y955C change, the most common human dominant mutation of POLG. The phenotypic characterization of POLG Y933C mice revealed disrupted mendelian distribution of offspring, indicating that the Y933C mutation is lethal when in homozygosity. However, the heterozygous mice did not display exercise intolerance or impaired motor coordination when compared to the WT littermates. Histological analysis of tissues from Polg+/Y933C showed no obvious morphological alterations in the brain, kidney, and heart at 5 months of age. However, at 24 months of age, the brain exhibited vacuolation and presence of activated microglial cells, indicating ongoing inflammation and neurodegeneration. Molecular analysis revealed that the Y933C mutation did not cause a significant reduction in mtDNA copy number in any of the tissues analyzed. However, in vitro characterization of the mutant recombinant POLA protein demonstrated severely impaired DNA synthesis for both mouse and human mutant proteins. Moreover, both mouse and human mutant proteins displayed a dominant negative effect on the replisome and could not support DNA synthesis on dsDNA templates.