Experimental Hematology Pharmacological inhibition of LSD1 and mTOR reduces mitochondrial retention and associated ROS levels in the red blood cells of sickle cell disease  Ramasamy Jagadeeswaran, Benjamin A. Vazquez, Muthusamy Thiruppathi, Balaji B. Ganesh, Vinzon Ibanez, Shuaiying Cui, James D. Engel, Alan M. Diamond, Robert E. Molokie, Joseph DeSimone, Donald Lavelle, Angela Rivers  Experimental Hematology  Volume 50, Pages 46-52 (June 2017) DOI: 10.1016/j.exphem.2017.02.003 Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 1 Mitochondria are retained in RBCs of SCD patients. (A) Whisker plot showing the percentage of mitochondria-containing RBCs in control (HbAA) and SCD (HbSS) mice. Center line in the plot represents the mean value. (B) Representative flow cytometry plots showing the percentage of cells in TO (RNA)-negative RBCs that retain mitochondria located in the upper- left quadrant in control and SCD mice. Experimental Hematology 2017 50, 46-52DOI: (10.1016/j.exphem.2017.02.003) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 2 Mitochondria are present in RBCs of humanized HbSS sickle mice and levels were associated with excessive ROS. RN-1 and sirolimus treatment reduced the level of abnormal mitochondria-retaining RBCs and decreased ROS levels. (A) Peripheral blood cells obtained from WT and SCD mice were stained with MitoTracker Green FM. (B) Peripheral blood cells obtained from WT and SCD mice or SCD mice treated with RN-1 (2.5 mg/kg), RN-1 (5 mg/kg), or sirolimus (5 mg/kg) were stained with TMRM and TO or CD71-APC-conjugated antibody. RBCs were gated based on specific forward and side scatter properties and analyzed by flow cytometry. The bar graph shows the mean percentage of peripheral mitochondria-retaining RBCs in WT versus SCD versus SCD treated with RN-1 (5 mg/kg) or sirolimus (5 mg/kg). (C) Representative flow cytometry plots showing the percentage of mitochondria-retaining RBCs located in the upper-left quadrant in WT, SCD, and SCD mice treated with RN-1 (5 mg/kg) or SCD mice treated with sirolimus (5 mg/kg). (D) Peripheral blood from control (HbAA) and SCD (HbSS) mice were incubated with the ROS probe CM-H2DCFDA after staining with TMRM and CD71-APC. The bar graph shows the mean percentage of RBCs with high ROS in WT versus SCD versus SCD treated with RN-1 (5 mg/kg) or sirolimus (5 mg/kg). (E) Representative flow cytometry plots showing the percentage of cells in the RBC fraction with ROS located in the upper-left quadrant in WT, SCD, and SCD mice treated with RN-1 (5 mg/kg) or sirolimus (5 mg/kg). (F) Pearson correlation plot showing the correlation between mitochondria and ROS levels in RBC of control and treated SCD mice. Experimental Hematology 2017 50, 46-52DOI: (10.1016/j.exphem.2017.02.003) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 3 Mitochondria present in RBCs of SCD mice analyzed by imaging flow cytometry. Representative images of cells (R3, R4, R5, R6) obtained by Image Stream X flow cytometer (Amnis). Gating strategy was used similar to regular flow cytometry detection of mitochondria-retaining RBCs methodology, as described in the Methods. The top left panel (R6) shows TO-negative mitochondria (TMRM-positive)-retaining RBCs. The bottom left panel (R3) shows TO-negative (TMRM-negative) RBCs that effectively eliminated mitochondria during terminal differentiation. The top right panel (R5) shows TO-positive mitochondria-retaining RBC precursors (reticulocytes). The bottom right panel (R4) shows TO-positive mitochondria-negative RBC precursors (reticulocytes). BF = Bright field. Experimental Hematology 2017 50, 46-52DOI: (10.1016/j.exphem.2017.02.003) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 4 RN-1 and sirolimus treatment increase RBC survival. The percentage of RBC survival was calculated by measuring the circulating biotin-labeled RBCs by staining with TER-119 PE and streptavidin AF488. The percentage reduction of labeled RBC was calculated relative to the first day. n = 2–3 of each type of mouse per group. Shown is the RBC survival curve of WT and SCD with PBS, RN-1 (2.5 mg/kg), RN-1 (5 mg/kg), or sirolimus (5 mg/kg) for 4 weeks. Each data point represents the mean ± SEM. Experimental Hematology 2017 50, 46-52DOI: (10.1016/j.exphem.2017.02.003) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 5 Mitophagy gene expression profiling of bone marrow cells from SCD mice treated with RN-1. (A) Heat map of 186 mitophagy-related genes that are differentially expressed (RN-1 treated vs. control) as determined by microarray analysis. Red denotes upregulation (fold changes). (B) Bar graph showing greater than twofold changes in upregulated mitophagy gene expression of bone marrow cells from SCD mice treated with RN-1 compared with vehicle-treated controls. Experimental Hematology 2017 50, 46-52DOI: (10.1016/j.exphem.2017.02.003) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 6 Schematic representation of the role of mitochondria-retaining RBCs in SCD pathogenesis. Mitochondria-retaining RBCs in SCD contribute excessive ROS, which destroy RBCs, leading to SCD pathogenesis. Inhibiting LSD1 by RN-1 or inhibiting mTOR by sirolimus reduces hemolysis by reducing mitochondria-retaining RBCs and associated ROS levels. Experimental Hematology 2017 50, 46-52DOI: (10.1016/j.exphem.2017.02.003) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions