Research ArticleGENETICS

Modulation of mtDNA copy number ameliorates the pathological consequences of a heteroplasmic mtDNA mutation in the mouse

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Science Advances  03 Apr 2019:
Vol. 5, no. 4, eaav9824
DOI: 10.1126/sciadv.aav9824
  • Fig. 1 Germ line transmission of the C5024T mutation and mtDNA copy number manipulation.

    (A) Schematic representation of mouse mtDNA and the structure of the mutated tRNAAla with a pathogenic mutation in the aminoacyl acceptor stem. (B) Quantification of mtDNA copy number by qPCR analysis (ND1/18S rRNA) of colon and heart from WT and C5024T mice at the age of 20 and 50 weeks. Data are represented as means ± SEM; n > 6. (C) Comparison of the levels of the C5024T mutation in offspring (n = 433) to heteroplasmic mothers (n = 28) measured in ear clips obtained at the age of 3 weeks. The dashed lines represent the maximum observed level of the mutation in the different mouse lines. (D) Western blot analysis of TFAM protein levels normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in colon and heart of mice with different genotypes at the age of 20 weeks. Data are represented as means ± SEM; n > 5. (E and F) Quantification of mtDNA copy number by qPCR (ND1/18S rRNA) in heart and colon at 20 (E) and 50 weeks of age (F). Animals with harboring WT or C5024T mtDNA combined with the Tfam+/+, Tfam+/OE, or the Tfam+/KO alleles were analyzed. Data are represented as means ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ns, nonsignificant; n > 6.

  • Fig. 2 Different levels of mtDNA modulate the phenotypic manifestations of the C5024T mutation.

    (A) The ratio of heart (HW) to body weight (BW) (g/g) in males at 50 weeks of age. (B) ANF and ATF5 expression levels as measured by qPCR at 50 weeks of age. Data are represented as means ± SEM; *P < 0.05; ***P < 0.001; n > 5. (C to D) Representative COX/SDH staining of colonic crypts and smooth muscle at 20 weeks of age (C) and of colonic crypts, smooth muscle, and heart at 50 weeks of age (D) from mice with WT or C5024T mtDNA combined with the Tfam+/+, Tfam+/OE, or the Tfam+/KO alleles. Scale bars, 100 μm (colonic crypt) 50 μm (heart and smooth muscle).

  • Fig. 3 Levels of mtDNA modulate tRNAAlaexpression, mitochondrial translation, and OXPHOS function.

    All analyses were performed in heart and colon of C5024T mice at 50 weeks of age. (A) Representative Northern blot analysis of mitochondrial tRNAAla and nuclear 5.8S rRNA (as loading control) levels. (B) Quantification of the steady-state levels of tRNAAla. (C) Representative in organello translation assays of mitochondria isolated from colon and heart. Coomassie staining and Western blot analysis of porin are shown as loading controls. CYTB, cytochrome b. (D) Respiratory chain enzyme activity assays of CI, CI + CIII, CII, CII + CIII, and CIV in mitochondrial protein extracts from heart and colon. Data are represented as means ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; n > 5.

  • Fig. 4 The mtDNA copy number regulates the segregation of the C5024T mutation in proliferating tissues of aged mice.

    (A to C) Relative levels of the C5024T mutation were measured with pyrosequencing. The mutation levels in an ear clip at 3 weeks of age was compared with mutation levels in the tail, quadriceps, muscle, heart, colon, and small intestine at 20 weeks (A), 50 weeks (B), and 75 weeks (C) of age in Tfam+/+ C5024T, Tfam+/OE C5024T, and Tfam+/KO C5024T mice. Data are represented as means ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; n > 10. (D to F) Comparison between levels of heteroplasmy in heart and colon of individual mice of the genotypes Tfam+/+ C5024T (D), Tfam+/OE C5024T (E), and Tfam+/KO C5024T (F) at 20, 50, and 75 weeks of age, respectively.

  • Fig. 5 The mtDNA copy number affects the levels of heteroplasmy in the colonic epithelium.

    (A) Representative COX/SDH staining of colonic epithelium. In white circles, two examples of COX+ (brown) and COX (blue) crypts collected with laser capture microdissection. (B and C) Levels of heteroplasmy measured in individual laser-captured crypts from Tfam+/+ C5024T, Tfam+/OE C5024T, and Tfam+/KO C5024T mice at 20 (B) and 50 (C) weeks of age. Data are represented as means ± SEM. Fifteen to 30 crypts (blue and brown) were collected from two mice for each genotype and time point; ***P < 0.001. (D) Schematic architecture of the colonic crypt. The bottom portion contains the stem cell compartment at the base of the crypt, whereas highly specialized and differentiated colon epithelial cells are localized in the upper (top) portion. (E) Levels of heteroplasmy measured in individual laser-captured bottom (circles) and top (triangles) segments of crypts from Tfam+/+ C5024T mice at 50 and 75 weeks of age and from Tfam+/KO C5024T mice at 50 weeks of age. Data are represented as a median ± interquartile range. In total, 21 to 28 half crypts (top and bottom) were collected from two mice for each genotype and time point. (F) Proposed model for clonal selection against high levels of the C5024T mutation in colonic crypts.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/4/eaav9824/DC1

    Fig. S1. Modulation of mtDNA levels in adult Tfam+/+ C5024T mice.

    Fig. S2. Tissue-specific effects of manipulation of mtDNA levels in WT and C5024T mice.

    Fig. S3. The copy number of mtDNA has a minor impact on the steady-state levels of OXPHOS subunits but does not influence the steady-state levels of mtDNA-encoded transcripts.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Modulation of mtDNA levels in adult Tfam+/+ C5024T mice.
    • Fig. S2. Tissue-specific effects of manipulation of mtDNA levels in WT and C5024T mice.
    • Fig. S3. The copy number of mtDNA has a minor impact on the steady-state levels of OXPHOS subunits but does not influence the steady-state levels of mtDNA-encoded transcripts.

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