Xiaoshan Zhou

Progressive loss of mitochondrial DNA in thymidine kinase 2-deficient mice

Deficient enzymatic activity of the mitochondrial deoxyribonucleoside kinases deoxyguanosine kinase (DGUOK) or thymidine kinase 2 (TK2) cause mitochondrial DNA (mtDNA)-depletion syndromes in humans. Here we report the generation of a Tk2-deficient mouse strain and show that the mice develop essentially normally for the first week but from then on exhibit growth retardation and die within 2-4 weeks of life. Several organs including skeletal muscle, heart, liver and spleen showed progressive loss of mtDNA without increased mtDNA mutations or structural alterations. There were no major histological changes in skeletal muscle, but heart muscle showed disorganized and damaged muscle fibers. Electron microscopy showed mitochondria with distorted cristae. The Tk2-deficient mice exhibited pronounced hypothermia and showed loss of hypodermal fat and abnormal brown adipose tissue. We conclude that Tk2 has a major role in supplying deoxyribonucleotides for mtDNA replication and that other pathways of deoxyribonucleotide synthesis cannot compensate for loss of this enzyme.

Loss of thymidine kinase 2 alters neuronal bioenergetics and leads to neurodegeneration

Mutations of thymidine kinase 2 (TK2), an essential component of the mitochondrial nucleotide salvage pathway, can give rise to mitochondrial DNA (mtDNA) depletion syndromes (MDS). These clinically heterogeneous disorders are characterized by severe reduction in mtDNA copy number in affected tissues and are associated with progressive myopathy, hepatopathy and/or encephalopathy, depending in part on the underlying nuclear genetic defect. Mutations of TK2 have previously been associated with an isolated myopathic form of MDS (OMIM 609560). However, more recently, neurological phenotypes have been demonstrated in patients carrying TK2 mutations, thus suggesting that loss of TK2 results in neuronal dysfunction. Here, we directly address the role of TK2 in neuronal homeostasis using a knockout mouse model. We demonstrate that in vivo loss of TK2 activity leads to a severe ataxic phenotype, accompanied by reduced mtDNA copy number and decreased steady-state levels of electron transport chain proteins in the brain. In TK2-deficient cerebellar neurons, these abnormalities are associated with impaired mitochondrial bioenergetic function, aberrant mitochondrial ultrastructure and degeneration of selected neuronal types. Overall, our findings demonstrate that TK2 deficiency leads to neuronal dysfunction in vivo, and have important implications for understanding the mechanisms of neurological impairment in MDS.

Thymidine Kinase 2 Deficiency-Induced mtDNA Depletion in Mouse Liver Leads to Defect β-Oxidation

Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2−/−) that progressively loses its mtDNA. The TK2−/− mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2−/− mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2−/− mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2−/− mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial β-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2−/− mice causes impaired mitochondrial function that leads to defect β-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies.

Transgene expression of Drosophila melanogaster nucleoside kinase reverses mitochondrial thymidine kinase 2 deficiency.

Background: Thymidine kinase 2 (TK2) deficiency causes severe mitochondrial DNA (mtDNA) depletion due to absence of nucleotides for mtDNA synthesis. Results: Nucleoside kinase from Drosophila melanogaster was able to rescue TK2-deficient mice. Conclusion: Nucleotide import into mitochondria can compensate the loss of TK2 in differentiated tissues. Significance: The results highlight mechanisms to be explored for treatment of mtDNA depletion. A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK+/− transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK+/−TK2−/− mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK+/−TK2−/− mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency.

Gene Expression Deregulation in Postnatal Skeletal Muscle of TK2 Deficient Mice Reveals a Lower Pool of Proliferating Myogenic Progenitor Cells

Loss of thymidine kinase 2 (TK2) causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA) depletion syndrome (MDS) in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue.

Hematopoiesis in the thymidine kinase 2 deficient mouse model of mitochondrial DNA depletion syndrome

Mitochondria are important for normal blood-cell development, and several diseases linked to mitochondrial DNA (mtDNA) show hematological manifestations. We recently generated a mouse strain deficient in expression of the mitochondrial pyrimidine nucleoside kinase thymidine kinase 2 (Tk2), showing that these mice exhibit progressive mtDNA depletion in multiple organs. We used this mouse strain as a model for mtDNA depletion syndromes to investigate the effects of mtDNA depletion on hematopoiesis. MtDNA levels in spleen from the Tk2-deficient mice were decreased 50%, but in contrast to all other investigated organs, both thymus and peripheral blood leukocytes showed normal mtDNA levels. Analysis of peripheral blood and cell populations in spleen, thymus, and bone marrow showed normal findings in the Tk2-deficient mice. The total rates of thymidine phosphorylation—which also include phosphorylation catalyzed by cytosolic Tk 1—in both spleen and thymus from wild-type mice were >50-fold higher than in liver, brain, and muscle. In summary, our data show that blood cells are less dependent on mitochondrial Tk2 compared with several other tissues and that these cells can synthesize deoxyribonucleotides required for mtDNA replication by alternative pathways such as phosphorylation of thymidine by cytosolic Tk1.

Long Term Expression of Drosophila melanogaster Nucleoside Kinase in Thymidine Kinase 2-deficient Mice with No Lethal Effects Caused by Nucleotide Pool Imbalances

Background: Expression of Drosophila melanogaster nucleoside kinase (Dm-dNK) in mice causes deoxyribonucleotide (dNTP) pool imbalances. Results: Long term Dm-dNK expression rescued thymidine kinase 2 (Tk2)-deficient mice without lethal side effects. Conclusion: Dm-dNK is a candidate to treat TK2 deficiency. Significance: The results highlight mechanisms involved in the in vivo regulation of dNTP pools. Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2−/−) mice extended the life span of Tk2−/− mice from 3 weeks to at least 20 months. The Dm-dNK+/−Tk2−/− mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK+/−Tk2−/− mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK+/− mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues.

Inhibition of glutamate oxaloacetate transaminase 1 in cancer cell lines results in altered metabolism with increased dependency of glucose

BackgroundGlutamate oxaloacetate transaminase 1 (GOT1) regulates cellular metabolism through coordinating the utilization of carbohydrates and amino acids to meet nutrient requirements. KRAS mutated cancer cells were recently shown to rely on GOT1 to support long-term cell proliferation. The aim of the present study was to address the role of GOT1 in the metabolic adaption of cancer cells.MethodsGOT1-null and knockdown cell lines were established through CRISPR/Cas9 and shRNA techniques. The growth properties, colony formation ability, autophagy and selected gene expression profiles were analysed. Glucose deprivation decreased the viability of the GOT1-null cells and rescue experiments were conducted with selected intermediates. The redox NADH/NAD+ homeostasis as well as lactate secretion were determined. GOT1 expression levels and correlation with survival rates were analysed in selected tumor databases.ResultsInhibition of GOT1 sensitized the cancer cells to glucose deprivation, which was partially counteracted by oxaloacetate and phosphoenol pyruvate, metabolic intermediates downstream of GOT1. Moreover, GOT1-null cells accumulated NADH and displayed a decreased ratio of NADH/NAD+ with nutrient depletion. The relevance of GOT1 as a potential target in cancer therapy was supported by a lung adenocarcinoma RNA-seq data set as well as the GEO:GSE database of metastatic melanoma where GOT1 expression was increased. High levels of GOT1 were further linked to poor survival as analysed by the GEPIA web tool, in thyroid and breast carcinoma and in lung adenocarcinoma.ConclusionsOur study suggests an important role of GOT1 to coordinate the glycolytic and the oxidative phosphorylation pathways in KRAS mutated cancer cells. GOT1 is crucial to provide oxaloacetate at low glucose levels, likely to maintain the redox homeostasis. Our data suggest GOT1 as a possible target in cancer therapy.

Metformin downregulates the mitochondrial carrier SLC25A10 in a glucose dependent manner

Graphical abstract Figure. No Caption available. Abstract Metformin, a commonly used agent in the treatment of type 2 diabetes, is also associated with reduced risk of cancer development and improvement in cancer survival. Although much is known about metformin, the mechanisms behind its anti‐cancer properties are not fully understood. In this study we addressed the role of a mitochondrial transporter commonly upregulated in cancer cells, SLC25A10, for cell survival and metabolism in the presence of metformin. SLC25A10 is a carrier in the mitochondrial inner membrane that transports malate and succinate out of the mitochondria, in exchange of phosphate and sulfate. We show that metformin treatment results in decreased gene expression of the SLC25A10 carrier both in lung cancer A549 mock cells and A549 SLC25A10 knockdown (siSLC25A10) cells. The decrease was even more pronounced when cells were grown at low glucose concentrations. The expression levels of key enzymes in glucose metabolism showed slightly altered mean values for all genes tested in both control cells and siSLC25A10 cells upon metformin treatment. The gene expression of the metabolic regulator glutamic‐oxaloacetic transaminase 1 decreased in wild type cells upon metformin treatment whereas there was a trend of increased expression in the siSLC25A10 cells upon metformin treatment. In addition, the gene expression of the cyclin‐dependent kinase inhibitor 1A was markedly increased in the siSLC25A10 compared to control A549 cells, and with even larger increases in the presence of metformin and at low glucose concentration. Our data show that in siSLC25A10 cell lines, metformin significantly alters the SLC25A10 carrier at both mRNA and protein levels and can thereby affect the supply of nutrients and the metabolic state of cancer cells.