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

Solid-phase Microextraction and Detection of Organophosphate Triesters in Indoor air

In the work underlying this thesis solid-phase microextraction (SPME) was evaluated as a passive sampling technique for organophosphate triesters in indoor air. These compounds are used on a large scale as flame-retarding and plastizicing additives in a variety of materials and products, and have proven to be common pollutants in indoor air. The main objective of this work was to develop an accurate method for measuring the volatile fraction. Such a method can be used in combination with active sampling to obtain information regarding the vapour/particulate distribution in different indoor environments. SPME was investigated under both equilibrium and non-equilibrium conditions and parameters associated with these different conditions were estimated. In Paper I, time-weighted average (TWA) SPME under dynamic conditions was investigated in order to obtain a fast air sampling method for organophosphate triesters. Among the investigated SPME coatings, the absorptive PDMS polymer had the highest affinity for the organophosphate triesters and was consequently used in all further work. Since the sampling rate is dependent on the agitation conditions, the linear airflow rates had to be carefully considered. Sampling periods as short as 1 hour were shown to be sufficient for measurements in the ng-μg m-3 range when using a PDMS 100-μm fibre and a linear flow rate above 7 cm s-1 over the fibre. SPME under equilibrium conditions is rather time-consuming, even under dynamic conditions, for slowly partitioning compounds such as organophosphate triesters. Nevertheless, this method has some significant advantages. For instance, the limit of detection is much lower compared to 1 h TWA sampling. Furthermore, the sampling time can be ignored as long as equilibrium has been attained. In Paper II, SPME under equilibrium conditions was investigated and evaluated for organophosphate triester vapours. Since temperature and humidity are closely associated with the distribution constant a simple study of the effect of these parameters was performed. The obtained distribution constants were used to determine the air levels in a common indoor environment. SPME and parallel active sampling on filters yielded similar results, indicating that the detected compounds were almost entirely associated with the vapour phase To apply dynamic SPME method in the field a sampler device, which enables controlled linear airflow rates to be applied, was constructed and evaluated (Paper III). This device was developed for application of SPME and active sampling in parallel. A GC/PICI-MS/MS method was developed and used in combination with active sampling of organophosphate triesters in indoor air (Paper IV). The combination of MS/MS and the soft ionization achieved with methanol as reagent gas yielded high selectivity and detection limits comparable to those provided by GC with nitrogen-phosphorus detection (NPD). The method limit of detection, when sampling 1.5 m3 of air, was in the range 0.1-1.4 ng m-3. In Paper V, the developed MS method was used in combination with SPME for indoor air measurements. The levels detected in the investigated indoor environments range from a few ng to μg m-3. Tris(2-chloropropyl) phosphate was detected at a concentration as high as 7 μg m-3 in a newly rebuilt lecture room

Hepatic expression of adipophilin, voltage dependent anion channel (VDAC) and cytochrome oxidase subunit II (COXII).

<p>Protein expression in livers of A) 7 days old and B) 12 days old TK2^+/+ and TK2^−/− mice detected with Western blot. Protein samples are from three individuals of each genotype and age. Bio-RAD Quantity one software was used to determine the intensity of the bands and statistical comparisons (two-tailed unpaired Student’s t-test) between TK2^+/+ and TK2^−/− were done. VDAC was used to normalize the data. Only 12 days old TK2^−/− mice exhibit 150% higher expression of adipophilin compared to TK2^+/+ mice (p≤0.05), no other difference was detected.</p

Transmission electron microscopy pictures of mitochondria in livers from TK2^+/+ and TK2^−/− F6 mice.

<p>Bars = 2 µm. Sections shown are representative of the samples.</p

Carnitine palmitoyltransferase activity in hepatic mitochondria from TK2^+/+ and TK2^−/− mice 14 days of age.

<p>The results represent data from four individuals of each genotype. Data presented as per cent activity (mean ± SEM) compared to TK2^+/+. The P-value for statistical comparison (two-tailed unpaired Student’s t-test) between TK2^+/+ and TK2^−/− is shown, **p≤0.01.</p

Mitochondrial ATP production rates standardized with citrate synthase activity.

<p>CS: citrate synthase; G: glutamate; M: malate; S: succinate; P: pyruvate; PC: palmitoyl-L-carnitine; R: rotenone. Mitochondrial CS activity was similar in the genotypes. For each time point data from 14 days old mice, three from each genotype, have been used (n = 3) and are presented as mean ± SEM. Statistical comparisons (two-tailed unpaired Student’s t-test) between TK2^+/+ and TK2^−/− were made but no statistical differences were detected.</p

Genes differentially expressed in liver tissue of 14 days old TK2^+/+ and TK2^−/− mice identified by a PCR-focused array.

<p>Data are expressed as mean fold change in TK2^−/− samples relative to TK2^+/+ control samples. The data analysis, including statistical comparisons, were done using Qiagen’s software RT² Profiler PCR Array Data Analysis version 3.5. Genes of TK2^−/− mice (n = 3) that differed by >2-fold compared with TK2^+/+ controls (n = 3) are included and highlighted in bold when p≤0.05.</p

Mitochondrial palmitate oxidation rate in liver homogenates of 14 days old TK2^+/+ and TK2^−/− mice.

<p>Three independent measurements were performed (TK2^+/+ n = 3, TK2^−/− n = 3) and comparisons between the genotypes were made within each measurement. Data presented as per cent activity (mean ± SEM) compared to TK2^+/+. The P-value for statistical comparison (two-tailed unpaired Student’s t-test) between TK2^+/+ and TK2^−/− is shown, *p≤0.05.</p

Serum levels of cholesterol and triglycerides in ApoB-containing, ApoA1-containing and total lipoprotein particles, as well as total serum nonesterified fatty acids (NEFA).

<p>For each time point data from at least four different mice have been used (n≥4) and are presented as mean ± SEM. The P-value for statistical comparison (two-tailed unpaired Student’s t-test) between TK2^+/+ and TK2^−/− of the same age group is shown, *p≤0.05, **p≤0.01.</p