Mitochondrial dysfunction and treatment strategies

The mitochondria are essential for cellular energy production and are involved in many processes in the cells. The mitochondria contain their own DNA (mtDNA) that is vital for oxidative phosphorylation since it encodes enzymes of the respiratory chain. Mutations in the mtDNA and alterations in the mtDNA copy number are attributed to various human disorders including cancer. Mitochondrial DNA depletion syndromes (MDS) are a heterogeneous group of disorders characterized by severe depletion of the mtDNA. MDS predominantly manifests in high energy demanding tissues such as the skeletal muscle, brain and liver. Mutations in the genes that are responsible for providing precursors for the mtDNA synthesis such as thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) are known to cause MDS.

In an attempt to rescue the mtDNA depletion caused by thymidine kinase 2 (Tk2) deficiency in mice, the deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in the Tk2 deficient mice (Dm-dNK+/-Tk2-/-). The Dm-dNK+/- expression was able to rescue the Tk2-/- mice and prolong their life span from 3 weeks to up to 20 months. The Dm-dNK expression driven by the CMV promoter was observed in all tissues with highest expression in skeletal muscle and lower expression in heart, liver and adipose tissues. Dm-dNK+/-Tk2-/- mice maintained normal mtDNA levels in the skeletal muscle and liver throughout the observation time of 20 months. The Dm-dNK expression resulted in highly elevated dNTP pools with dTTP pools being >100 times higher than in the wild type mice. However, the large increase in the dTTP pool did not cause mutations in the nuclear or the mitochondrial DNA. A significant reduction in total body fat (both subcutaneous and visceral fat) was observed only in the Dm-dNK+/-Tk2-/- mice compared to wild type mice, which indicates an altered fat metabolism in these mice mediated through residual Tk2 deficiency.

To elucidate effective treatment strategies for TK2 deficiency, a novel mouse model with liver specific expression of Dm-dNK driven by the albumin promoter was generated. Two founder mice with high Dm-dNK expression and activity in the liver was selected for further characterization. These mice will be used to study whether Dm-dNK expression in a single tissue would be able to rescue the sever phenotype caused by Tk2 deficiency in mice.

The mitochondrial dicarboxylate carrier, SLC25A10, is involved in the transport of dicarboxylates such as malate and succinate across the mitochondrial inner membrane. To understand the role of the SLC25A10 carrier in regulating cancer cell growth, metabolism and transformation, a knockdown of SLC25A10 in a lung adenocarcinoma cell line (A549) was established and characterized. The growth properties of SLC25A10 knockdown cells changed to a less malignant phenotype, with increased dependency on glutamine and altered NADPH production. An increase in expression of glutamate dehydrogenase and decrease in expression of lactate dehydrogenase indicated a metabolic shift from glycolysis to oxidative phosphorylation in the SLC25A10 knockdown cells. The study demonstrates the importance of SLC25A10 in and regulation of redox homeostasis.

List of scientific papers

I. Shuba Krishnan, Xiaoshan Zhou, João A. Parades, Raoul, V. Kuiper, Sophie Curbo, Anna Karlsson. Transgene Expression of Drosophila melanogaster Nucleoside Kinase Reverses Thymidine Kinase 2 Deficiency. J Biol Chem. 2013 Feb 15; 288(7):5072-5079.
https://doi.org/10.1074/jbc.M112.437152

II. Shuba Krishnan, João A. Parades, Xiaoshan Zhou, Raoul, V. Kuiper, Kjell Hultenby, Sophie Curbo, Anna Karlsson. Long Term Expression of Drosophila melanogaster Nucleoside Kinase in Thymidine Kinase 2-deficient Mice with no Lethal Effects Caused by Nucleotide Pool Imbalance. J Biol Chem. 2014 Nov 21; 289(47):32835-32844.
https://doi.org/10.1074/jbc.M114.588921

III. Shuba Krishnan, Xiaoshan Zhou, Sophie Curbo, Anna Karlsson. Construction of a mouse strain with liver specific expression of Drosophila melanogaster nucleoside kinase. [Manuscript]

IV. Xiaoshan Zhou, João A. Paredes, Shuba Krishnan, Sophie Curbo, Anna Karlsson. The mitochondrial carrier SLC25A10 regulates cancer cell growth. Oncotarget. 2015 Apr 20; 6(11):9271-9283.
https://doi.org/10.18632/oncotarget.3375