Thymidine kinase 2 deficiency-induced mtDNA depletion in mouse liver leads to defect β-oxidation

Abstract

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.

Figure 1. Transmission electron microscopy pictures of mitochondria in livers from TK2^+/+ and TK2^−/− F6 mice.

Bars = 2 µm. Sections shown are representative of the samples.

Figure 2. Hepatic expression of adipophilin, voltage dependent anion channel (VDAC) and cytochrome oxidase subunit II (COXII).

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.

Figure 3. Hepatic gene expression and lipid content.

A) Hepatic cholesteryl esters in 7 and 12 days old TK2^+/+ and TK2^−/− mice determined with GC-MS. B) Relative hepatic mRNA expression of 3-hydroxy-3-methyl-glutaryl-Coenzyme A reductase (Hmgcr) and 3-hydroxy-3-methyl-glutaryl-Coenzyme A synthase (Hmgcs) in 7 and 12 days old TK2^+/+ and TK2^−/− mice determined with real-time PCR. C) Hepatic triglycerides in 7, 10 and 12 days old TK2^+/+ and TK2^−/− mice determined with an enzymatic assay. D) Relative hepatic mRNA expression of fatty acid synthase (Fas) and sterol regulatory element binding protein 1c (Srebp1c) in 7 and 12 days old TK2^+/+ and TK2^−/− mice determined with real-time PCR. Data presented as mean ± SEM. Statistically significant difference (two-tailed unpaired Student’s t-test) compared to TK2^+/+ of the same age, *p≤0.05.

Figure 4. Mitochondrial palmitate oxidation rate in liver homogenates of 14 days old TK2^+/+ and TK2^−/− mice.

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.

Figure 5. Carnitine palmitoyltransferase activity in hepatic mitochondria from TK2^+/+ and TK2^−/− mice 14 days of age.

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.