Genetics of Mitochondrial Diseases
Medical Research Foundation
Venetian Institute of Molecular Medicine
In “Mitofight” we are engaged in developing suitable therapeutic approaches against mitochondrial disorders in mouse models with a particular but not exclusive emphasis on Pearson syndrome and related conditions associated with instability of mtDNA. The creation of mouse models has to be followed by their characterisation in vivo and ex vivo, and eventually, to the evaluation of any beneficial effect induced by therapeutic strategies. In order to accomplish these goals Massimo Zeviani started his laboratory at VIMM dedicated to the investigation of our mouse disease models both in their behaviour (locomotor proficiency, orientation in space, cognitive functions) and after their culling (to study pathological changes in different tissues). In the frame of this project, we have characterized a knockin mouse reproducing the most common recessive mutation in the mitochondrial DNA Polymerase (POLGA467T). We demonstrated that the mutant POLG has severely impaired DNA binding, polymerase, and proofreading activities. In addition, mutant protein has reduced affinity for the accessory subunit POLG2, which makes POLG amenable of degradation by the LONP1 protease. This study highlights a new mechanism contributing to the pathogenesis of POLG-related disorders. In addition, we have produced a Y955C mouse model reproducing the most common dominant mutation of the patients. The initial characterization of this model showed that the mutant homozygous mice were embryonic lethal with complete penetrance. In contrast, the heterozygous did not show any obvious clinical phenotype. Post-mortem analysis showed only a partial increase of mtDNA content in skeletal muscle and liver in KI vs WT littermates. Older animals (around two years of age) showed spongiosis in several brain areas and inflammatory infiltrates in skeletal muscle. No major phenotypes were observed in other organs, such as liver, kidney and heart. No mtDNA 2 depletion or deletions were observed in any organ. In order to trigger a more severe phenotype, we challenged two-year old Y933C mice with a high-fat, ketogenic diet, for two months. No differences were observed between wild-type and mutant animals. We also tried to challenge the animals by administering AZT, a nucleoside analogue which causes mtDNA depletion in humans, in the drinking water, but again, we did not observe any obvious effect on the clinical phenotype or on mtDNA amount/quality. In collaboration with Maria Falkenberg, University of Gothenburg, we investigated the molecular pathogenesis of the mouse Y933C and humanY955C mutations in vitro. The data are clearcut and show that both mutant proteins have a DNA binding capacity similar to the wildtype counterparts, but that capacity to incorporate nucleotides is severely impaired. In addition, we demonstrated that the mutant protein has a dominant effect and inhibits the wild-type form. Future studies will be aimed at using additional challenges to trigger a more severe phenotype (eg: inducing fever by LPS administration) and in developing gene editing approaches to correct the mutation.
2.1 Biological and endogenous factors