Mitophagy Induction Is a Potential Therapeutic Approach for Sickle Cell Disease

Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide. A single point mutation of the sixth amino acid of β-globin causes glutamic acid to be replaced by valine, rendering the hemoglobin susceptible to polymerization when deoxygenated. SCD patients suffer from the wide variety of disease manifestations including chronic hemolytic anemia, inflammation, painful vaso-occlusive crises, multisystem organ damage, and reduced life expectancy. In addition to the HbS polymerization-mediated rigid and fragile sickle-shaped red blood cell (RBC) formation, an excessive formation of intracellular reactive oxygen species (ROS) occurs in SCD red blood cells, which accelerates their hemolysis. This causes the release of ROS, free extracellular hemoglobin, hemin, and inflammatory cytokines that trigger disease progression. We analyzed levels of ROS in SCD patient RBCs and observed a higher fraction of intracellular ROS positive RBC in SCD (HbSS) compared to control (HbAA) RBC of adults [Control (HbAA): 7.1%± 1.4 %, n=11; SCD (HbSS): 25.3 % ± 4.3%, n=9; p<0.0004]. We also made the novel observation that mature RBCs from SCD patients abnormally contain mitochondria as evidenced by flow cytometry analysis of blood samples of 36 SCD patients and 14 normal human control subjects.[Control (HbAA):0.4 % ± 0.04%, n=14; SCD (HbSS): 7.8%± 0.9%, n=30; p<0.0001]. Further subset analysis from SCD patients with HbSC showed mitochondrial retention in their mature RBCs [HbSC: 2.2%± 0.6%,n=6 p<0.01], however to a lesser degree than patients with HbSS. Transmission electron microscopy confirmed the presence of mitochondria in mature RBC of patients with SCD. ROS analysis between mitochondria positive vs. negative fractions showed that mitochondria-positive (TMRM+) RBC fractions have higher levels of ROS compared to mitochondria-negative (TMRM-) RBC fractions. This data strongly suggests that retained mitochondria significantly contribute to the production of ROS in SCD RBCs. Similar to humans, a higher fraction of RBCs of SCD mice (B6;129-Hba^tm1(HBA)Tow Hbb^{tm2(HBG1,HBB*)Tow/J}) retain mitochondria compared to control mice RBC [Control (HbAA): 0.29% ± 0.18%; SCD (HbSS): 16.68%± 1.9%, p<0.0001]. While investigating RN-1, a lysine specific demethylase-1 (LSD-1) inhibitor, as a HbF inducing agent, we observed that SCD mice treated with RN-1 showed a reduction in the fraction of RBCs which retain mitochondria. Therefore, we investigated mitophagy-inducing drugs as a possible useful therapeutic approach for SCD by administering mitophagy-inducing agent Sirolimus. SCD mice treated with RN-1 (5mg), or Sirolimus (5mg) had a significant decrease in the fraction of mitochondria containing RBCs (RN1: 4.96± 1.0%, p<0.0005; Sirolimus: 6.4% ± 1.8%, p<0.002). We observed a reduction of ROS in mature RBCs coupled with decreased mitochondrial retention in RBCs after in vivo treatment with RN1 or Sirolimus as measured by co-staining of TMRM, APC-conjugated CD71antibody, and CM-H2DCFDA. We also observed a significant improvement in RBC survival after the in vivo treatment with Sirolimus or RN-1. RBC survival was measured by flow cytometry and calculated biotin positive circulating RBCs after 2 days of in vivo labeling [SCD treated with vehicle control: 40 %± 2.6%; SCD treated with RN1 (2.5mg): 69.9 ± 2.6%, p<0.004; Sirolimus (5mg): 67.5% ± 6.1%, p<0.04]. Based on this data, mitophagy-inducing drugs have the potential to be a novel therapeutic approach for the treatment of SCD patients.