Universität zu Köln

Sendon, Pamella Marie (2024). Development and characterization of a novel mouse model carrying mitochondrial DNA mutations. PhD thesis, Universität zu Köln.

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Abstract

Mitochondria perform vital functions in the cell by regulating bioenergetics, metabolism, and signaling pathways. These organelles contain their own DNA, known as the mitochondrial DNA (mtDNA) which encodes mitochondrial ribosomal RNAs, transfer RNAs, and messenger RNAs constituting some of the subunits of oxidative phosphorylation (OXPHOS). Pathogenic mtDNA mutations can therefore affect the OXPHOS structural subunits or the mitochondrial protein synthesis machinery resulting in the development of mitochondrial diseases. In this regard, animal models are a valuable tool in understanding the mechanisms underlying mtDNA mutations and mitochondrial diseases in vivo. The research presented in this thesis aims to identify specific mtDNA mutations and their molecular and biochemical consequences, investigate the effect of these mutations on the mouse phenotype, and compare the novel mouse model to the previously characterized C5024T. In this work, we generated a novel mouse model through a breeding strategy utilizing female heterozygous mutator mice with PolgA proofreading deficiency. We identified three mtDNA point mutations in a single mtDNA molecule cosegregating with mitochondrial deficiency: m.5019AG, m.15200AG, and m.16232AT. The m.15200A>G mutation is a synonymous mutation located in the Cytochrome b gene while the m.16232AT mutation caused a very little reduction in transcription initiation according to in vitro transcription assay; however, this has no effect on mt-mRNA steady-state levels and mtDNA copy number in vivo. The m.5019AG mutation does not affect mt-tRNAALA stability but impacts mt-tRNAALA aminoacylation as shown by the presence of deacylated mt-tRNAALA molecules in the aminoacylation assay. We then refer to this novel mouse model as A5019G mice. Phenotypically, A5019G male mice exhibited a lean phenotype with age; however, we observed no sign of heart enlargement associated with cardiomyopathy in A5019G mice, unlike in C5024T mice. Tissue-specific COX deficiency occurs in A5019G mice with the intestinal epithelial tissue being impacted the most while rare occurrence of COX deficiency is found in skeletal muscle and lung tissues. Proteomics revealed extensive changes occurring in the intestinal epithelium proteome of A5019G mice as they age. Furthermore, analysis of the mitochondrial proteins in these mice showed a significant reduction in the expression of the majority of Complex I and Complex IV protein subunits with upregulation of proteins for OXPHOS assembly, mtRNA metabolism, and mitochondrial translation as a compensatory mechanism. In conclusion, the major pathogenic mutation causing mitochondrial dysfunction in this novel mouse model is the m.5019AG mutation which affects the aminoacylation of mt-tRNAALA resulting in difficulties in translation of protein subunits of Complexes I and IV. Accordingly, the work covered in this thesis offers valuable insights into the control and regulation of mtDNA heteroplasmy and mitochondrial dysfunction. This work can be used in future studies involving mitochondrial genome engineering techniques to reduce mtDNA heteroplasmy and rescue the animals from OXPHOS defects, which is pertinent for potential therapeutic approaches for mitochondrial diseases.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Sendon, Pamella Marie pmdsendon@gmail.com UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-736845
Date:	2024
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	Natural sciences and mathematics
Uncontrolled Keywords:	Keywords Language mouse model English mitochondria English mitochondrial DNA English
Date of oral exam:	29 May 2024
Referee:	Name Academic Title Trifunovic, Aleksandra Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/73684