Polymorphism nt7778G/T In Mitochondrial ATP8 Gene Promotes Protective Effect On Reactive Oxygen Species Level In Murine Hematopoietic Cells During Aging

Mitochondria are complex cell compartments characterized by a nuclear genome independent own genome referred to as mitochondrial genome (mtDNA). MtDNA encodes 13 proteins which are part of the five enzyme complexes of the mitochondrial respiratory chain. The respiratory chain is responsible for ATP synthesis and is the main source of reactive oxygen species (ROS) in the cell. During cellular aging, mutations in mtDNA accumulate leading potentially to respiratory chain deficiency. Due to the lack of DNA repair mechanisms within the mitochondrion itself, the mtDNA is especially vulnerable towards ROS. Until now it is not fully understood whether intracellular ROS levels increase with aging in all cell types, and whether this process is due to a potential impaired function of the respiratory chain. Thus, the influence of mtDNA mutations on oxidative stress and aging of hematopoietic cells remains to be investigated. Herein, we compared two conplastic mouse strains differing in one point mutation in the mtDNA affecting the respiratory chain. The C5BL/6Ntac-mt^FVB/NJ (mtFVB) strain carries a point mutation at nt7778G/T leading to a D>Y replacement in ATP8 protein, while the control stain C5BL/6Ntac-mt^AKR (mtAKR) does not. The targeted protein is part of the F₀sub unit of complex V in the respiratory chain and we hypothesize that the mutation affects the transport of protons within the complex V.

In our study, we analyzed bone marrow cells of both strains at four different aging stages (3 to 24 months) for ROS and ATP levels by DCFH-fluorescence and luminescence staining, respectively. Additionally, the subpopulations of bone marrow cells were analyzed by flow cytometric immunophenotyping. Further, blood counts of the mice treated with a single dose of 5 Fluorouracil (5-FU, 150mg/kg BW, i.p.) were performed every 3 days during a total time span of 21 days.

The mtAKR strain showed increasing levels of ROS ranging from 7.1 x 10³ to 12 x 10³ relative fluorescence units (RFU) and decreasing levels of ATP (from 0.99 to 0.36 µM) during the measurement time points 3 to 24 months. In comparison, the mtFVB strain showed a significant decrease in ROS levels ranging from 5.5 x 10³ to 2.4 x 10³ RFU and a significant increase of ATP levels (from 0.23 to 0.64 µM) during the same period. Hematopoietic cells of aged mtFVB mice (24 months) contained significantly more ATP than cells of mtAKR mice. Analysis of bone marrow cell composition of both strains showed an increase of hematopoietic stem cells (LSK cells: lineage-, sca-1+, c-kit+) during experimental time span from 3 to 24 months (mtAKR: from 2.13 to 2.81 % of lineage^- cells, mtFVB: from 2.13 to 3.52 %). However, only in the mtFVB strain this increase was significant. Interestingly, the amount of LSK cells was significant higher in mtFVB compared to the mtAKR strain (mtFVB: 2.36 %, mtAKR: 1.23 %) at 6 months. Furthermore, erythroide cells (Ter119⁺) in mtAKR increased with aging (from 25.22 % to 32.69 %), while the mtFVB strain showed a decreased rate (from 25.88 % to 18.9 %).

Blood counts of 3 months old mtFVB mice treated with 5-FU were similar to those of the mtAKR strain in the myelosuppression phase after application, but demonstrated higher regeneration peaks at day 15 in white blood cells (mtAKR: 20.24 x 10³/µl, mtFVB: 25.72 x 10³/µl), platelets (mtAKR: 2016 x 10³/µl, mtFVB: 2848 x 10³/µl), neutrophilic (mtAKR: 0.8 x 10³/µl, mtFVB: 2.36 x 10³/µl) and eosinophilic granulocytes (mtAKR: 0.28 x 10³/µl, mtFVB: 0.76 x 10³/µl) and lymphocytes (mtAKR: 17.12 x 10³/µl, mtFVB: 21.84 x 10³/µl).

The mtDNA polymorphism in complex V of the respiratory chain in mtFVB strain influences the development of ROS and ATP levels in hematopoietic cells during aging, and seems to have a protective impact concerning ROS and ATP levels in aged mice. Furthermore, the capability of LSK cells to expand with age seems to be enhanced in mtFVB strain. Moreover, the regeneration capacity after 5-FU myelosuppression seems also to be increased in 3 months old mice.