Cathepsin B, a Negative Regulator of Autophagy, Identified As a Novel Therapeutic Drug Target in Sickle Cell Disease

Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide. The disease is caused by a mutation of the beta-globin gene that results in polymerization of the sickle hemoglobin (HbS) when deoxygenated. Reactive oxygen species (ROS) induced hemolysis is a major critical event in SCD. We have previously shown that abnormal retention of mitochondria in the erythrocytes of both SCD patients and SCD mice is associated with elevated ROS levels and hemolysis (Exp. Hem.2017;50:46-52). The mechanism responsible for mitochondrial retention in SCD is unknown. Autophagy is one of the processes responsible for the elimination of mitochondria during erythroid differentiation. Autophagy is a conserved physiological process that promotes cellular homeostasis through the recycling of proteins, protein aggregates, and removal of damaged organelles. Hypothesis: In this study, we have investigated the hypothesis that autophagy pathway dysregulation is responsible for abnormal mitochondrial retention in SCD erythrocytes. We propose that pharmacological methods that address the abnormal activity of these genes may be therapeutic for SCD. Methods: The expression of autophagy pathway genes in reticulocytes of SCD patients and experimental SCD mice was compared with their respective controls using the RT2 Autophagy Profiler PCR Array. The expression of autophagy proteins was measured by Western blot. Cathepsin B (CTSB) activity was measured by a fluorometric assay using the substrate RR-AFC (amino-4-trifluoromethyl coumarin). Results: Six autophagy pathway genes were differentially expressed in SCD patients compared to control subjects. Expression of the autophagy pathway genes was also analyzed in SCD mice, normal AA mice, and phlebotomized anemic AA mice as a control for increased reticulocytosis. Eighteen genes were differentially expressed in SCD mice compared with control AA mice (p<0.05). Four genes were differentially expressed in SCD mice compared with control phlebotomized anemic AA mice (p<0.05). CTSB, a known negative regulator of autophagy (J Exp Med. 2016;213(10):2081-97), was over-expressed in the reticulocytes of both SCD patients and SCD mice. Expression of CTSB was 4.5 fold (p<0.007) higher in SCD mice compared to normal control AA mice and 8.6 fold (p<0.001) higher compared to phlebotomized AA anemic mice. We therefore focused our further analysis on CTSB. Western blot data confirmed that CTSB protein was overexpressed in red blood cells of SCD patients and experimental SCD mice (p<0.01) compared to respective controls. The level of CTSB enzyme activity was 3.3 fold higher in SCD mice compared to the control mice(p<0.01). To evaluate the functional significance of these differences in CTSB expression, CTSB inhibitor E64d was investigated in SCD mice. SCD mice treated with E64d (40mg/kg b.wt.) for two weeks showed a reduction in CTSB activity (SCD vehicle: 11.8±1.1 RFU/µg, n=3 vs SCD E64d treatment: 5 ± 0.1 RFU/µg, n=3, p<0.008) and in the proportion of reticulocytes that retained mitochondria (SCD vehicle: 45.2%±3%, n=3 vs. SCD E64d treatment: 32 % ± 1.9%, n=3, p<0.003). Reticulocytosis was also significantly reduced in SCD mice treated with the CTSB inhibitor (SCD vehicle: 47.2%±2.8%, n=3 vs. SCD E64d treatment: 35.9 % ± 3.9%, n=3, p<0.01). Conclusion: Our data shows that the multiple autophagy pathway gene expression is dysregulated in SCD. Among these genes, CTSB, a key negative regulator of autophagy, is overexpressed in the reticulocytes of both SCD patients and SCD mice. Pharmacological inhibition of CTSB activity partially reverses mitochondrial retention and decreases reticulocytosis in SCD mice, confirming that overexpression of CTSB contributes to SCD pathogenesis and is a potential therapeutic target.