SOD2-deficiency anemia: protein oxidation and altered protein expression reveal targets of damage, stress response, and antioxidant responsiveness by Jeffrey S. Friedman, Mary F. Lopez, Mark D. Fleming, Alicia Rivera, Florent M. Martin, Megan L. Welsh, Ashleigh Boyd, Susan R. Doctrow, and Steven J. Burakoff Blood Volume 104(8):2565-2573 October 15, 2004 ©2004 by American Society of Hematology

Iron deposition and morphology on EM of SOD2-deficient red cells. Iron deposition and morphology on EM of SOD2-deficient red cells. (A) Modified Perl stain of peripheral blood from recipients of Sod2+/+ or Sod2–/– fetal liver transplants (as a source of hematopoietic stem cells) demonstrating excess iron on SOD2-deficient cells. (B) Electron micrographs of Sod2+/+ and Sod2–/– reticulocytes reveal an increased number of mitochondria with prominent intramitochondrial membranes in SOD2-deficient cells. No obvious foci of iron deposition (electrondense material) were seen within SOD2-deficient cells with standard EM processing. (C) Electron micrographs of reticulocytes prestained with potassium ferricyanide prior to processing. Small arrows show diffuse electron-dense deposits in matrix of mitochondria, and large arrows show strong electron-dense deposits in the outer mitochondrial membrane of Sod2–/– cells. Bar length is 500 nm. Jeffrey S. Friedman et al. Blood 2004;104:2565-2573 ©2004 by American Society of Hematology

Zinc protoporphyrin levels. Zinc protoporphyrin levels. ZPP levels are higher in Sod2–/– peripheral red cells compared with Sod2+/+. Treatment of SOD2-deficient (transplant recipient) mice with the antioxidant EUK-189 (duration 4 weeks) leads to a further increase in ZPP levels. The increase occurs despite a concomitant increase in hematocrit (from 0.237 ± 0.02 [23.7% ± 2%] without treatment to 0.305 ± 0.03 [30.5% ± 3%] after therapy) and a decrease in reticulocyte count (from 0.175 ± 0.06 [17.5% ± 6%] prior to therapy to 0.072 ± 0.007 [7.2% ± 0.7%] after therapy). Data presented are mean ± standard deviation; ANOVA was used for statistical analysis with Tukey-Kramer multiple comparison test between the 3 groups (n = 5 animals per group). In a second experiment (right), the effect of Euk-189 on Sod2+/+ red cells was assessed following therapy for 21 days (n = 4 animals per group). Although these data are suggestive of an effect of Euk-189 on ZPP in Sod2+/+ cells, results from this trial were not statistically significant (2-tailed t test, P = .07). Jeffrey S. Friedman et al. Blood 2004;104:2565-2573 ©2004 by American Society of Hematology

Sod2+/+ and Sod2–/– spleen cells stained with H&E. Sod2+/+ and Sod2–/– spleen cells stained with H&E. Erythroid hyperplasia is evident in SOD2-deficient samples. Occasional nuclear clefting is evident in Sod2–/– erythroid precursors (arrow), suggesting mild dysplasia. Jeffrey S. Friedman et al. Blood 2004;104:2565-2573 ©2004 by American Society of Hematology

Protein oxidation in marrow progenitors and peripheral red cells. Protein oxidation in marrow progenitors and peripheral red cells. Protein carbonyl content in Sod2+/+ and Sod2–/– samples was compared by Oxyblot. Lane 1 (Sod2+/+) and lane 2 (Sod2–/–) compare whole-cell lysate protein from 5 × 106 Ter119+ marrow progenitor cells isolated by FACS. Lane 3 (Sod2+/+), lane 4 (Sod2–/–), and lane 5 (Sod2–/– Euk-189–treated) contain 20 μg red cell cytosolic protein, and lane 6 (Sod2+/+), lane 7 (Sod2–/–), and lane 8 (Sod2–/– Euk-189–treated) contain 5 μg membrane protein from the corresponding red cell samples. Jeffrey S. Friedman et al. Blood 2004;104:2565-2573 ©2004 by American Society of Hematology

Effect of antioxidant therapy on red cell lifespan. Effect of antioxidant therapy on red cell lifespan. Mice that received transplants of Sod2–/– fetal liver stem cells were divided into 4 groups for measurement of red cell lifespan in the presence or absence of SOD/catalase antioxidant therapy. Group A was pretreated with antioxidant for 4 weeks and continued treatment during measurement of red cell lifespan. Group B was pretreated as Group A, but stopped antioxidant at the time of red cell labeling with biotin. Group C commenced Euk-189 therapy coincident with red cell labeling, whereas group D received sham injections. Error bars = SD. Jeffrey S. Friedman et al. Blood 2004;104:2565-2573 ©2004 by American Society of Hematology

ROS generation by reticulocytes and mature red cells. ROS generation by reticulocytes and mature red cells. Peripheral blood cells from Sod2+/+ and Sod2–/– transplant recipients were incubated in the presence of the superoxide-sensitive dye DHE and the peroxide-sensitive dye H2DCFDA for 30 minutes at 37° C, followed by FACS analysis. (Left) Peroxide production by Sod2+/+ red cells and reticulocytes (shaded gray); and Sod2–/– red cells and reticulocytes (black line). (Right) Superoxide production by Sod2+/+ (shaded gray) and Sod2–/– (black line) red cells and reticulocytes. Red cells are the major peak for each histogram, with reticulocytes forming a tail or secondary peak with higher ROS production. Plots shown are representative of 5 mice for each genotype. For each histogram: light gray tracing indicates unstained Sod2+/+ cells; broken black tracing indicates unstained Sod2–/– cells. Jeffrey S. Friedman et al. Blood 2004;104:2565-2573 ©2004 by American Society of Hematology

Representative 2-D gel analysis of membrane and soluble protein preparations from Sod2+/+, Sod2–/–, and Euk-189–treated Sod2–/– red cells. Representative 2-D gel analysis of membrane and soluble protein preparations from Sod2+/+, Sod2–/–, and Euk-189–treated Sod2–/– red cells. Membrane (200 μg) or soluble red cell protein (400 μg) was separated by 2-D gel electrophoresis. Sod2+/+ red cell samples were obtained from serially phlebotomized animals (in order to induce reticulocytosis similar to that seen in Sod2–/– samples). Each sample was prepared from pooled red cells obtained from 5 donor animals. Jeffrey S. Friedman et al. Blood 2004;104:2565-2573 ©2004 by American Society of Hematology