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Mitochondrial DNA with a large-scale deletion causes two distinct mitochondrial disease phenotypes in mice.

Katada S, Mito T, Ogasawara E, Hayashi J, Nakada K - G3 (Bethesda) (2013)

Bottom Line: Late-stage embryos carrying ≥50% ΔmtDNA showed abnormal hematopoiesis and iron metabolism in livers that were partly similar to PS (PS-like phenotypes), although they did not express sideroblastic anemia that is a typical symptom of PS.More than half of the neonates with PS-like phenotypes died by 1 month after birth, whereas the rest showed a decrease of ΔmtDNA load in the affected tissues, peripheral blood and liver, and they recovered from PS-like phenotypes.The proportion of ΔmtDNA in various tissues of the surviving mito-miceΔ increased with time, and Kearns-Sayre syndrome-like phenotypes were expressed when the proportion of mtDNA in various tissues reached >70-80%.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life and Environmental Sciences, International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japan.

ABSTRACT
Studies in patients have suggested that the clinical phenotypes of some mitochondrial diseases might transit from one disease to another (e.g., Pearson syndrome [PS] to Kearns-Sayre syndrome) in single individuals carrying mitochondrial (mt) DNA with a common deletion (ΔmtDNA), but there is no direct experimental evidence for this. To determine whether ΔmtDNA has the pathologic potential to induce multiple mitochondrial disease phenotypes, we used trans-mitochondrial mice with a heteroplasmic state of wild-type mtDNA and ΔmtDNA (mito-miceΔ). Late-stage embryos carrying ≥50% ΔmtDNA showed abnormal hematopoiesis and iron metabolism in livers that were partly similar to PS (PS-like phenotypes), although they did not express sideroblastic anemia that is a typical symptom of PS. More than half of the neonates with PS-like phenotypes died by 1 month after birth, whereas the rest showed a decrease of ΔmtDNA load in the affected tissues, peripheral blood and liver, and they recovered from PS-like phenotypes. The proportion of ΔmtDNA in various tissues of the surviving mito-miceΔ increased with time, and Kearns-Sayre syndrome-like phenotypes were expressed when the proportion of mtDNA in various tissues reached >70-80%. Our model mouse study clearly showed that a single ΔmtDNA was responsible for at least two distinct disease phenotypes at different ages and suggested that the level and dynamics of mtDNA load in affected tissues would be important for the onset and transition of mitochondrial disease phenotypes in mice.

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Related in: MedlinePlus

Phenotypic observations of late-stage embryos of mito-mice∆. All embryos were obtained at embryonic day 18.5. (A) Morphological observation of late-stage embryos carrying 0–66% ∆mtDNA in their tails, as indicated. The embryo carrying no ∆mtDNA (left side) was a wild-type B6 control. The six embryos carrying ∆mtDNA were obtained from a single mother. The weight of each embryo is shown below the panel. (B) Comparison of littermate number. Average numbers of littermates from mothers carrying 0% (white), 1–32% (gray), or 33–63% (black) ∆mtDNA. Data are presented as mean ± SD. NS, not significant. (C) Relationship between weight and ∆mtDNA load in late-stage embryos carrying 0% (white), 34–47% (gray), or 52–66% (black) ∆mtDNA. Data are presented as mean ± SD. Asterisks indicate significant differences (P < 0.05). (D) Cytological observation of peripheral blood samples from late-stage embryos. Peripheral blood samples carrying 0%, 36%, and 59% ∆mtDNA, respectively, were stained with new methylene blue. In this method, reticulocytes, a type of immature red blood cells (RBC), are visualized as cells with blue inclusions (open arrowheads). Scale bar, 10 μm. (E) Average proportion of reticulocytes in total RBCs in peripheral blood samples from late-stage embryos carrying 0% (white), 34–42% (gray), or 52–65% (black) ∆mtDNA. Data are presented as mean ± SD. Asterisks indicate significant differences (P < 0.05). (F) Histologic observations of iron metabolism in liver samples from late-stage embryos. Liver samples carrying 0%, 41%, and 55% ∆mtDNA, respectively, were stained with Prussian blue. In this method, abnormal iron metabolism is visualized as blue deposits. Scale bar, 10 μm. (G) and (H) Electron microscopic observation of COX activity in liver samples from late-stage embryos. Mitochondria in hepatocytes and blood cells are shown as (G) and (H), respectively. Arrows indicate mitochondria that appeared normal in shape and COX activity, open arrowheads indicate COX-deficient mitochondria, and the closed arrowheads indicate swollen mitochondria with COX activity. N, nucleus. Scale bar, 1 μm.
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fig3: Phenotypic observations of late-stage embryos of mito-mice∆. All embryos were obtained at embryonic day 18.5. (A) Morphological observation of late-stage embryos carrying 0–66% ∆mtDNA in their tails, as indicated. The embryo carrying no ∆mtDNA (left side) was a wild-type B6 control. The six embryos carrying ∆mtDNA were obtained from a single mother. The weight of each embryo is shown below the panel. (B) Comparison of littermate number. Average numbers of littermates from mothers carrying 0% (white), 1–32% (gray), or 33–63% (black) ∆mtDNA. Data are presented as mean ± SD. NS, not significant. (C) Relationship between weight and ∆mtDNA load in late-stage embryos carrying 0% (white), 34–47% (gray), or 52–66% (black) ∆mtDNA. Data are presented as mean ± SD. Asterisks indicate significant differences (P < 0.05). (D) Cytological observation of peripheral blood samples from late-stage embryos. Peripheral blood samples carrying 0%, 36%, and 59% ∆mtDNA, respectively, were stained with new methylene blue. In this method, reticulocytes, a type of immature red blood cells (RBC), are visualized as cells with blue inclusions (open arrowheads). Scale bar, 10 μm. (E) Average proportion of reticulocytes in total RBCs in peripheral blood samples from late-stage embryos carrying 0% (white), 34–42% (gray), or 52–65% (black) ∆mtDNA. Data are presented as mean ± SD. Asterisks indicate significant differences (P < 0.05). (F) Histologic observations of iron metabolism in liver samples from late-stage embryos. Liver samples carrying 0%, 41%, and 55% ∆mtDNA, respectively, were stained with Prussian blue. In this method, abnormal iron metabolism is visualized as blue deposits. Scale bar, 10 μm. (G) and (H) Electron microscopic observation of COX activity in liver samples from late-stage embryos. Mitochondria in hepatocytes and blood cells are shown as (G) and (H), respectively. Arrows indicate mitochondria that appeared normal in shape and COX activity, open arrowheads indicate COX-deficient mitochondria, and the closed arrowheads indicate swollen mitochondria with COX activity. N, nucleus. Scale bar, 1 μm.

Mentions: Blood samples were smeared on glass slides, and inclusions in reticulocytes were visualized by staining with new methylene blue. Cells with granular network inclusions that are a typical structure of reticulocytes were counted (see open arrowheads in Figure 3D) and proportion of these reticulocytes to 1000 red blood cells was estimated. The proportion of reticulocytes in blood samples of mito-mice∆ and age-matched controls was measured. To detect the accumulation of ferric iron, paraffin-embedded sections (5-μm thick) of liver samples were stained with Prussian blue and then Safranin O was used as a counter-staining. Eye samples were fixed in Bouin’s solution, and paraffin-embedded sections (8-μm thick) of the samples were stained with hematoxylin and eosin.


Mitochondrial DNA with a large-scale deletion causes two distinct mitochondrial disease phenotypes in mice.

Katada S, Mito T, Ogasawara E, Hayashi J, Nakada K - G3 (Bethesda) (2013)

Phenotypic observations of late-stage embryos of mito-mice∆. All embryos were obtained at embryonic day 18.5. (A) Morphological observation of late-stage embryos carrying 0–66% ∆mtDNA in their tails, as indicated. The embryo carrying no ∆mtDNA (left side) was a wild-type B6 control. The six embryos carrying ∆mtDNA were obtained from a single mother. The weight of each embryo is shown below the panel. (B) Comparison of littermate number. Average numbers of littermates from mothers carrying 0% (white), 1–32% (gray), or 33–63% (black) ∆mtDNA. Data are presented as mean ± SD. NS, not significant. (C) Relationship between weight and ∆mtDNA load in late-stage embryos carrying 0% (white), 34–47% (gray), or 52–66% (black) ∆mtDNA. Data are presented as mean ± SD. Asterisks indicate significant differences (P < 0.05). (D) Cytological observation of peripheral blood samples from late-stage embryos. Peripheral blood samples carrying 0%, 36%, and 59% ∆mtDNA, respectively, were stained with new methylene blue. In this method, reticulocytes, a type of immature red blood cells (RBC), are visualized as cells with blue inclusions (open arrowheads). Scale bar, 10 μm. (E) Average proportion of reticulocytes in total RBCs in peripheral blood samples from late-stage embryos carrying 0% (white), 34–42% (gray), or 52–65% (black) ∆mtDNA. Data are presented as mean ± SD. Asterisks indicate significant differences (P < 0.05). (F) Histologic observations of iron metabolism in liver samples from late-stage embryos. Liver samples carrying 0%, 41%, and 55% ∆mtDNA, respectively, were stained with Prussian blue. In this method, abnormal iron metabolism is visualized as blue deposits. Scale bar, 10 μm. (G) and (H) Electron microscopic observation of COX activity in liver samples from late-stage embryos. Mitochondria in hepatocytes and blood cells are shown as (G) and (H), respectively. Arrows indicate mitochondria that appeared normal in shape and COX activity, open arrowheads indicate COX-deficient mitochondria, and the closed arrowheads indicate swollen mitochondria with COX activity. N, nucleus. Scale bar, 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3755915&req=5

fig3: Phenotypic observations of late-stage embryos of mito-mice∆. All embryos were obtained at embryonic day 18.5. (A) Morphological observation of late-stage embryos carrying 0–66% ∆mtDNA in their tails, as indicated. The embryo carrying no ∆mtDNA (left side) was a wild-type B6 control. The six embryos carrying ∆mtDNA were obtained from a single mother. The weight of each embryo is shown below the panel. (B) Comparison of littermate number. Average numbers of littermates from mothers carrying 0% (white), 1–32% (gray), or 33–63% (black) ∆mtDNA. Data are presented as mean ± SD. NS, not significant. (C) Relationship between weight and ∆mtDNA load in late-stage embryos carrying 0% (white), 34–47% (gray), or 52–66% (black) ∆mtDNA. Data are presented as mean ± SD. Asterisks indicate significant differences (P < 0.05). (D) Cytological observation of peripheral blood samples from late-stage embryos. Peripheral blood samples carrying 0%, 36%, and 59% ∆mtDNA, respectively, were stained with new methylene blue. In this method, reticulocytes, a type of immature red blood cells (RBC), are visualized as cells with blue inclusions (open arrowheads). Scale bar, 10 μm. (E) Average proportion of reticulocytes in total RBCs in peripheral blood samples from late-stage embryos carrying 0% (white), 34–42% (gray), or 52–65% (black) ∆mtDNA. Data are presented as mean ± SD. Asterisks indicate significant differences (P < 0.05). (F) Histologic observations of iron metabolism in liver samples from late-stage embryos. Liver samples carrying 0%, 41%, and 55% ∆mtDNA, respectively, were stained with Prussian blue. In this method, abnormal iron metabolism is visualized as blue deposits. Scale bar, 10 μm. (G) and (H) Electron microscopic observation of COX activity in liver samples from late-stage embryos. Mitochondria in hepatocytes and blood cells are shown as (G) and (H), respectively. Arrows indicate mitochondria that appeared normal in shape and COX activity, open arrowheads indicate COX-deficient mitochondria, and the closed arrowheads indicate swollen mitochondria with COX activity. N, nucleus. Scale bar, 1 μm.
Mentions: Blood samples were smeared on glass slides, and inclusions in reticulocytes were visualized by staining with new methylene blue. Cells with granular network inclusions that are a typical structure of reticulocytes were counted (see open arrowheads in Figure 3D) and proportion of these reticulocytes to 1000 red blood cells was estimated. The proportion of reticulocytes in blood samples of mito-mice∆ and age-matched controls was measured. To detect the accumulation of ferric iron, paraffin-embedded sections (5-μm thick) of liver samples were stained with Prussian blue and then Safranin O was used as a counter-staining. Eye samples were fixed in Bouin’s solution, and paraffin-embedded sections (8-μm thick) of the samples were stained with hematoxylin and eosin.

Bottom Line: Late-stage embryos carrying ≥50% ΔmtDNA showed abnormal hematopoiesis and iron metabolism in livers that were partly similar to PS (PS-like phenotypes), although they did not express sideroblastic anemia that is a typical symptom of PS.More than half of the neonates with PS-like phenotypes died by 1 month after birth, whereas the rest showed a decrease of ΔmtDNA load in the affected tissues, peripheral blood and liver, and they recovered from PS-like phenotypes.The proportion of ΔmtDNA in various tissues of the surviving mito-miceΔ increased with time, and Kearns-Sayre syndrome-like phenotypes were expressed when the proportion of mtDNA in various tissues reached >70-80%.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life and Environmental Sciences, International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japan.

ABSTRACT
Studies in patients have suggested that the clinical phenotypes of some mitochondrial diseases might transit from one disease to another (e.g., Pearson syndrome [PS] to Kearns-Sayre syndrome) in single individuals carrying mitochondrial (mt) DNA with a common deletion (ΔmtDNA), but there is no direct experimental evidence for this. To determine whether ΔmtDNA has the pathologic potential to induce multiple mitochondrial disease phenotypes, we used trans-mitochondrial mice with a heteroplasmic state of wild-type mtDNA and ΔmtDNA (mito-miceΔ). Late-stage embryos carrying ≥50% ΔmtDNA showed abnormal hematopoiesis and iron metabolism in livers that were partly similar to PS (PS-like phenotypes), although they did not express sideroblastic anemia that is a typical symptom of PS. More than half of the neonates with PS-like phenotypes died by 1 month after birth, whereas the rest showed a decrease of ΔmtDNA load in the affected tissues, peripheral blood and liver, and they recovered from PS-like phenotypes. The proportion of ΔmtDNA in various tissues of the surviving mito-miceΔ increased with time, and Kearns-Sayre syndrome-like phenotypes were expressed when the proportion of mtDNA in various tissues reached >70-80%. Our model mouse study clearly showed that a single ΔmtDNA was responsible for at least two distinct disease phenotypes at different ages and suggested that the level and dynamics of mtDNA load in affected tissues would be important for the onset and transition of mitochondrial disease phenotypes in mice.

Show MeSH
Related in: MedlinePlus