Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance

Abstract

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2(-/-)) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2(-/-) animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype.

Figure 1.

Tk2^−/− mice show growth retardation and early mortality. (A) Body weight of the mice with increasing age. Squares represent wild-type Tk2^+/+ mice (n = 16); circles, Tk2^+/− mice and triangles, Tk2^−/− mice. (B) Kaplan–Meier curves showing percentages of surviving mice at the indicated ages. Squares represent wild-type Tk2^+/+ mice (n = 30) and triangles, 10 Tk2^−/− mice (n = 10).

Figure 2.

Tk2^−/− mice show reduced locomotor activity on open-field testing. (A) Representative 15 min open-field test of 12-day-old mice Tk2^+/+ and Tk2^−/− littermates. (B) Average distance walked (cm) by 12-day-old Tk2^+/+ (n = 4) and Tk2^−/− (n = 6) mice, *P < 0.002.

Figure 3.

Tk2^−/− mice have reduced Tk2 activity in multiple tissues. Tk2 activity expressed as percent of the global TK activity (Tk1+Tk2) in Tk2^−/− and wild-type mice. The experiments were performed in duplicate of whole extracts. Data are expressed as mean±SD of n = 3, **P<0.01 versus Tk2^+/+.

Figure 4.

Tk2-deficiency alters mitochondrial dNTP pools. Levels of dATP, dTTP, dCTP and dGTP in mitochondria from mouse brain (A) and liver (B). All mice were sacrificed between 12 and 18 days old. Data are expressed as mean±SD of n = 5, ***P<0.001 versus Tk2^+/+.

Figure 5.

Tk2−/− mice show depletion of mtDNA in multiple tissues. Tk2^−/− mice showed partial depletion of mitochondrial DNA in all tissues tested (in decreasing order of severity brain, spinal cord, heart, muscle, kidney and liver). Data are expressed as mean±SD of n = 5. *P<0.05 versus Tk2^+/+, **P < 0.01 versus Tk2^+/+, ***P < 0.001 versus Tk2^+/+.

Figure 6.

Tk2^−/− mice have decreased activities of respiratory chain enzymes and reduced steady-state levels of mtDNA-encoded proteins in brain, but not visceral organs or muscle. (A) Brain of Tk2^−/− mice showed decreased activities of mitochondrial complexes I and IV normalized to citrate synthase and expressed in terms of percent of activity in wild-type mice. (B) Reduced levels of mtDNA-encoded subunits subunit 6 of NADH dehydrogenase (complex I) and subunit I of cytochrome c oxidase (complex IV) in brain were detected by western blot performed with whole tissue protein extracts (30 µg/lane) from three 12–14-day-old Tk2^+/+ (lanes 1–3) and three Tk2^−/− (lanes 4–6) mice using Rodent Total OXPHOS Complexes Detection Kit cocktail of antibodies (MitoSciences, Eugene, OR, USA). Western blots performed with other tissues under the same conditions are shown in supplemental data (Supplementary Material, Fig. S2). All mice were sacrificed between ages 12 and 18 days. Data are expressed as mean±SD of five mice per group. **P < 0.01 versus Tk2^+/+, CI, complex I; CIV, complex IV; CS, citrate synthase.

Figure 7.

Histological studies show vacuolar changes and gliosis in spinal cord and gliosis in brain of Tk2^−/− mice. Hematoxylin and eosin (H–E) staining of wild-type (A) and Tk2^−/− brain (B) and wild-type spinal cord (E) appeared normal in contrast to the Tk2^−/− anterior spinal cord (F) which showed vacuolar changes in neurons (arrows). Anti-glial fibrillary acid protein (anti-GFAP) antibody staining of wild-type spinal cord (G) and brain (C) showed normal astrocytes while Tk2^−/− mice show abnormal and striking gliotic changes evident as large numbers of GFAP immunoreactive astrocytes with large cell bodies and processes in both Tk2^−/− brain (D) and spinal cord (H).