Kauppila, Johanna Heta Katariina (2018). Generating mammalian mitochondrial disease models with mitochondrial DNA mutations. PhD thesis, Universität zu Köln.


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Mitochondria are vitally important cellular organelles that are instrumental to many cellular functions such as energy conversion, iron-sulfur cluster synthesis and -oxidation of fatty acids. Although, the vast majority of mitochondrial proteins are encoded by the nuclear DNA and transported into mitochondria post-translationally, mitochondria also contain their own DNA, mitochondrial DNA (mtDNA), which is essential for mitochondrial function. Mammalian mtDNA encodes 2 rRNAs and 22 tRNAs that are required to translate the 11 protein-coding mRNAs encoded by mtDNA. The encoded proteins are essential components of the oxidative phosphorylation system (OXPHOS) and therefore pathogenic mtDNA mutations can lead to drastic energy deficiency disorders with typically pleiotropic symptoms including progressive neurodegeneration, muscle weakness, epilepsy, stroke and different type of myopathies. Despite extensive research, the genotype-phenotype correlations and tissue specificity of mitochondrial disorders remain still an enigma. Comprehensive molecular understanding of these diseases has been hindered by the limited number of animal models available for research. Because mtDNA cannot be efficiently modified with molecular-biology techniques, the main strategy to generate these animal models has been a to introduce mutations found in cell lines into mouse ES cells. In this thesis, two genetic approaches are presented to introduce endogenous mutations to mtDNA. These strategies utilized both natural sources of mtDNA mutations, namely replication errors and oxidative damage to mtDNA. In the first approach, proofreading-deficient DNA polymerase  is utilized to mutate mtDNA and in the second approach, mitochondrial DNA repair is impaired to increase the prevalence of oxidative stress driven mutations. The repair is impaired by abolishing the mitochondrial localization of two base-excision repair glycosylases, OGG1 and MUTYH. In the first approach, maternal lineages carrying limited number of mtDNA mutations were generated by breeding the heterozygous proofreading deficient female mice (mtDNA mutator mice) for one generation. Next, the induced pathogenic mtDNA mutations were identified via a new strategy, by screening the colonic crypts of the founder mice for respiratory chain dysfunction. The mtDNA mutations can rapidly clonally expand in colonic crypts to reach heteroplasmy levels high enough to induce OXPHOS dysfunction, which allows a straight-forward and early detection of mouse lineages that carry pathogenic mtDNA mutations. With this approach, a founder mouse was identified that carried a C5024T mutation in mitochondrial tRNAALA gene. These tRNAALA mice display typical molecular phenotypes seen in classical mitochondrial diseases, i.e. decreased stability of the mutated tRNAALA transcript, impaired mitochondrial translation and presence of respiratory chain deficient cells. In summary, the results show that heterozygous mtDNA mutator mice in combination with colonic-crypt screening for pathogenic mutations, is a successful approach to generate mouse models for mitochondrial research. Surprisingly, the second mutagenic approach, using dysfunctional base-excision repair (BER) in mitochondria, did not result into an increase in mtDNA mutation load even when the mice were aged. As DNA repair is suggested to be especially important in the germ line and the BER deficient mice were therefore bred for five consecutive generation as a homozygote maternal line. However, no increase mtDNA mutation load was detected also in these mice. To increase prevalence of oxidative stress in these animals they were bred with tissue specific superoxide dismutase 2 (SOD2) knockout mice. The heart Sod2 knockout mice show a clear increase in superoxide levels demonstrated by loss of aconitase activity and a plethora of changes in mitochondrial function. However, no increase in mtDNA or mtRNA mutation load was detected in the repair deficient heart Sod2 knockout mice. These results demonstrate that firstly BER deficiency is not a feasible approach to introduce mutations to mtDNA and secondly that the importance of oxidative stress as a contributor to mtDNA mutation load should be re-evaluated. Instead, in both ageing research and mitochondrial disease models, we should focus on replication errors as the source of mtDNA mutations.

Item Type: Thesis (PhD thesis)
CreatorsEmailORCIDORCID Put Code
Kauppila, Johanna Heta Katariinajohanna.kauppila@age.mpg.deUNSPECIFIEDUNSPECIFIED
URN: urn:nbn:de:hbz:38-84765
Date: April 2018
Language: English
Faculty: Faculty of Mathematics and Natural Sciences
Divisions: Außeruniversitäre Forschungseinrichtungen > MPI for Biology of Ageing
Subjects: Life sciences
Uncontrolled Keywords:
oxidative stressUNSPECIFIED
mitochondrial diseaseUNSPECIFIED
Date of oral exam: 20 June 2018
NameAcademic Title
Larsson, Nils-GöranProf. Dr.
Trifunovic, AleksandraProf. Dr.
Riemer, JanProf. Dr.
Smeitink, JanProf. Dr.
Refereed: Yes
URI: http://kups.ub.uni-koeln.de/id/eprint/8476


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