Research ArticleTHALASSEMIA

The autophagy-activating kinase ULK1 mediates clearance of free α-globin in β-thalassemia

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Science Translational Medicine  21 Aug 2019:
Vol. 11, Issue 506, eaav4881
DOI: 10.1126/scitranslmed.aav4881
  • Fig. 1 Ablation of Ulk1 in hematopoietic stem cells exacerbates β-thalassemia.

    (A) Kaplan-Meier survival plot of mice with germline genotypes Hbb+/+Ulk1+/+ (n = 30), Hbb+/+Ulk1−/− (n = 20), HbbTh3/+Ulk1+/+ (n = 20), and HbbTh3/+Ulk1−/− (n = 15). (B) Embryonic E14.5 fetal liver cells with the genotypes in (A) (C57BL/6, CD45.2) were injected intravenously into lethally irradiated WT (Hbb+/+Ulk1+/+) mice (C57BL/6, CD45.1). (C) Erythroid indices (y axis) according to donor HSC genotype (x axis) at 90 days after HSC transplantation. Hbb+/+Ulk1+/+, n = 22; HbbTh3/+Ulk1+/+, n = 29; HbbTh3/+Ulk1−/−, n = 25. RBC, red blood cell number; Hb, hemoglobin; Retic, reticulocyte. (D) RBC survival at time 0 (about 90 days after HSC transplantation), sulfo-NHS-biotin was injected into the tail vein of mice with HSCs of the indicated genotypes. The fraction of biotinylated RBCs over time was quantified by streptavidin labeling followed by flow cytometry: Hbb+/+Ulk1+/+, n = 8; HbbTh3/+Ulk1+/+, n = 7; HbbTh3/+Ulk1−/−, n = 4. Differences between HbbTh3/+Ulk1+/+ and HbbTh3/+Ulk1−/− mice were statistically significant at all times measured between days 7 and 25 (false discovery rate–adjusted P = 0.002 to 0.02 by the Benjamini and Hochberg method). (E) Representative spleens from mice transplanted with HSC donors of the indicated genotypes. Scale bars, 0.5 cm. (F) Summary of spleen weights: Hbb+/+Ulk1+/+, n = 19; HbbTh3/+Ulk1+/+, n = 15; HbbTh3/+Ulk1−/−, n = 12. Data are mean values ± SDs; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant.

  • Fig. 2 ULK1 loss in β-thalassemia increases accumulation of insoluble α-globin.

    WT mice were transplanted with fetal liver HSCs as in Fig. 1B and analyzed 90 to 120 days later. (A) Soluble and insoluble globins in RBCs. Equal volumes of RBCs (normalized by hematocrit) were lysed, centrifuged to separate insoluble and soluble proteins, fractionated by TAU gel electrophoresis to resolve α- and β-globin proteins, and stained with Coomassie brilliant blue. (B) Results of multiple experiments performed as in (A). The y axes represent the relative soluble and insoluble α-globin staining intensities on TAU gels measured by automated image analysis and expressed in arbitrary units. Hbb+/+Ulk1+/+, n = 4; HbbTh3/+Ulk1+/+, n = 11; HbbTh3/+Ulk1−/−, n = 8. (C) Thiazole orange–stained reticulocytes were isolated by fluorescence-activated cell sorting (FACS) to a purity of about 95% and then analyzed by TEM. AI, α-globin inclusions; MT, mitochondria. Scale bars, 1 μm. (D) Areas of electron-dense α-globin inclusions in reticulocytes (y axis) determined by automated image analysis of electron micrographs. Hbb+/+Ulk1+/+, n = 7; HbbTh3/+Ulk1+/+, n = 11; HbbTh3/+Ulk1−/−, n = 12. About 100 to 200 cells from each mouse were analyzed. (E) Western blot analysis of autophagy proteins ULK1 and p62 in FACS-purified reticulocytes from mice with the indicated genotypes. Migration of protein molecular weight standards is shown on the right. (F) Results of multiple experiments performed as in (E). The y axis represents the relative protein staining intensity on Western blots, measured by automated image analysis, normalized to β-actin, and shown in arbitrary units: n = 3 mice for each genotype. Data are mean values ± SDs; ***P < 0.001; *P < 0.05; ns, not significant.

  • Fig. 3 Clearance of free α-globin in β-thalassemia is largely ATG5 independent.

    (A) Erythroid indices (y axis) of mice with the indicated germline genotypes (x axis) at 5 months of age: Hbb+/+Atg5+/+, n = 10; HbbTh3/+Atg5+/+, n = 10; HbbTh3/+Atg5fl/fl, n = 12. The Atg5 fl allele refers to an erythroid-specific targeted disruption of Atg5 (fig. S5). (B) Spleen weight according to genotype. Hbb+/+Atg5+/+, n = 10; HbbTh3/+Atg5+/+, n = 5; HbbTh3/+Atg5fl/fl, n = 4. (C) Soluble and insoluble α-globin in RBCs determined as described for Fig. 2A. (D) Results of multiple experiments performed as in (C) and quantified as described for Fig. 2B. Hbb+/+Atg5+/+, n = 5; HbbTh3/+Atg5+/+, n = 8; HbbTh3/+Atg5fl/fl, n = 8. (E) Western blot analysis of LC3 in FACS-purified reticulocytes cultured for 3 hours with chloroquine (CQ) to inhibit lysosomal proteases or with vehicle (Veh). Migration of protein molecular weight standards is shown on the right. (F) Results of multiple experiments performed as in (E), measured by automated image analysis, normalized to β-actin, and shown in arbitrary units: n = 4 mice for each genotype. Data are mean values ± SDs; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant.

  • Fig. 4 Lysosome-dependent flux of insoluble free α-globin in reticulocytes is ATG5 independent and ULK1 dependent.

    (A) Reticulocytes in whole blood from mice with the indicated genotypes were pulse-labeled with 35S-amino acids and then chased with unlabeled amino acids ± a proteasome inhibitor [MG132 (MG), 10 μM] and/or lysosome inhibitor (chloroquine, 100 μM). Insoluble α-globin in cell lysates was resolved by TAU gel electrophoresis and then visualized by autoradiography. (B) Results of multiple experiments performed as in (A), quantified as described for Fig. 2B: HbbTh3/+, n = 8; HbbTh3/+Ulk1−/−, n = 5; HbbTh3/+Atg5fl/fl, n = 4. Data are mean values ± SDs; ****P < 0.0001; ***P < 0.001; **P < 0.01; ns, not significant versus vehicle for each genotype, by two-way analysis of variance (ANOVA).

  • Fig. 5 Rapamycin acts through ULK1 to alleviate clinical phenotypes of β-thalassemia in mice.

    WT mice were transplanted with fetal liver HSCs of the indicated genotypes, as in Fig. 1B. Twelve weeks later, rapamycin (Rap; 4 mg/kg) or vehicle was administered intraperitoneally, once daily for 30 days. (A) Protein synthesis rates in splenic and bone marrow erythroblasts, determined by O-propargyl-puromycin incorporation. Ery.A, Ery.B, and Ery.C represent increasingly mature Ter119+ erythroblast populations designated according to forward scatter and CD71 expression (see fig. S3): vehicle, n = 3; rapamycin, n = 4. (B) Erythroid indices (y axis) according to HSC genotype (x axis) after treatment with rapamycin or vehicle. HbbTh3/+Ulk1+/+, n = 15; HbbTh3/+Ulk1−/−, n = 12. (C) Sulfo-NHS-biotin was injected intravenously on day 13 of rapamycin treatment, and the fraction of biotinylated RBCs was quantified serially by streptavidin labeling and flow cytometry. (D) Spleens of β-thalassemic mice (HbbTh3/+Ulk1+/+) after rapamycin or vehicle treatment. Scale bars, 0.5 cm. (E) Summary of spleen weights in multiple HbbTh3/+Ulk1+/+ mice after treatment with rapamycin (n = 12) or vehicle (n = 12). Data are mean values ± SDs; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant.

  • Fig. 6 Rapamycin induces ULK1-dependent elimination of free α-globin.

    (A) Relative concentrations of insoluble α-globin in RBCs after 30-day treatment with rapamycin or vehicle in mice of the indicated genotypes. RBCs were analyzed as described in Fig. 2A. (B) Results of multiple experiments performed as in (A) and quantified as described for Fig. 2B. n = 7 mice for each condition shown. (C) Transmission electron micrographs of purified reticulocytes in mice with HSCs of the indicated genotypes treated with rapamycin or vehicle. Scale bars, 2 μm. (D) Areas of electron-dense α-globin inclusions in reticulocytes (y axis) determined by analysis of electron micrographs, as described for Fig. 2D. HbbTh3/+Ulk1+/+ + Rap, n = 7; HbbTh3/+Ulk1+/+ + Veh, n = 8; HbbTh3/+Ulk1−/− + Rap, n = 7; HbbTh3/+Ulk1−/− + Veh, n = 7. (E) Western blot analysis of LC3 and p62 in reticulocytes from mice with HSCs of the indicated genotypes treated with rapamycin or vehicle. Migration of protein molecular weight standards is shown on the right. (F) Results of multiple experiments performed as in (E). The y axis represents the relative protein staining intensity on Western blots, measured by automated image analysis, normalized to β-actin, and shown in arbitrary units: n = 4 mice for each genotype. Data are mean values ± SDs; ****P < 0.0001; ***P < 0.001; **P < 0.01; ns, not significant.

  • Fig. 7 Rapamycin reduces free α-globin in CD34+ cell–derived erythroblasts from individuals with β-thalassemia.

    CD34+ cells from normal donors (n = 7) or individuals with transfusion-dependent (TD; n = 5) or non–transfusion-dependent (NTD; n = 5) β-thalassemia were grown in culture under conditions that promote erythroid differentiation, treated with rapamycin (10 or 20 μM), MG132 (2.5 μM), or vehicle beginning on day 13 for 2 days, and then analyzed for free α-globin by ion exchange (IE)–HPLC. (A) Representative chromatograms. UV abs, ultraviolet absorbance. (B) Quantification of free α-globin fractions in multiple experiments performed as in (A). Data are mean values ± SDs; ****P < 0.0001; ***P < 0.001; **P < 0.01.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/506/eaav4881/DC1

    Materials and Methods

    Fig. S1. Ablation of Ulk1 in hematopoietic cells.

    Fig. S2. Phenotyping of ablation of Ulk1 in hematopoietic cells.

    Fig. S3. Developmental staging of erythroblasts in spleen and bone marrow of HbbTh3/+Ulk1+/+ and HbbTh3/+Ulk1−/− mice.

    Fig. S4. Characteristics of autophagy in β-thalassemic RBC precursors.

    Fig. S5. Ablation of Atg5 in erythroid cells.

    Fig. S6. Elimination of insoluble α-globin by the proteasome and autophagy in β-thalassemic reticulocytes.

    Fig. S7. Effects of systemic rapamycin on β-thalassemic erythropoiesis.

    Fig. S8. Expression of Map1lc3bm (LC3) and Sqstm1 (p62) mRNAs in reticulocytes of HbbTh3/+Ulk1+/+ mice treated with rapamycin.

    Fig. S9. Presence of free α-globin in CD34+ cell–derived erythroblasts from individuals with β-thalassemia.

    Fig. S10. Rapamycin and MG132 treatment of CD34+ cells from normal donors or from individuals with β-thalassemia.

    Fig. S11. Model for ULK1 kinase–dependent clearance of free α-globin in β-thalassemia.

    Table S1. Effects of Ulk1 gene disruption on erythroid indices of β-thalassemic mice.

    Table S2. Effects of Ulk1 loss on erythroid hyperplasia in β-thalassemic mice.

    Table S3. Effects of Atg5 gene disruption on erythroid indices of β-thalassemic mice.

    Table S4. Effects of systemic rapamycin on erythroid indices of β-thalassemic mice ± Ulk1 gene disruption.

    Table S5. ULK1-dependent reduction of erythroid hyperplasia in β-thalassemic mice treated with rapamycin.

    Table S6. Erythroid indices of individuals with β-thalassemia who provided CD34+ cells for this study.

    Table S7. Genotypes of individuals with β-thalassemia who provided CD34+ cells for this study.

    References (7276)

  • This PDF file includes:

    • Materials and Methods
    • Fig. S1. Ablation of Ulk1 in hematopoietic cells.
    • Fig. S2. Phenotyping of ablation of Ulk1 in hematopoietic cells.
    • Fig. S3. Developmental staging of erythroblasts in spleen and bone marrow of HbbTh3/+Ulk1+/+ and HbbTh3/+Ulk1−/− mice.
    • Fig. S4. Characteristics of autophagy in β-thalassemic RBC precursors.
    • Fig. S5. Ablation of Atg5 in erythroid cells.
    • Fig. S6. Elimination of insoluble α-globin by the proteasome and autophagy in β-thalassemic reticulocytes.
    • Fig. S7. Effects of systemic rapamycin on β-thalassemic erythropoiesis.
    • Fig. S8. Expression of Map1lc3bm (LC3) and Sqstm1 (p62) mRNAs in reticulocytes of HbbTh3/+Ulk1+/+ mice treated with rapamycin.
    • Fig. S9. Presence of free α-globin in CD34+ cell–derived erythroblasts from individuals with β-thalassemia.
    • Fig. S10. Rapamycin and MG132 treatment of CD34+ cells from normal donors or from individuals with β-thalassemia.
    • Fig. S11. Model for ULK1 kinase–dependent clearance of free α-globin in β-thalassemia.
    • Table S1. Effects of Ulk1 gene disruption on erythroid indices of β-thalassemic mice.
    • Table S2. Effects of Ulk1 loss on erythroid hyperplasia in β-thalassemic mice.
    • Table S3. Effects of Atg5 gene disruption on erythroid indices of β-thalassemic mice.
    • Table S4. Effects of systemic rapamycin on erythroid indices of β-thalassemic mice ± Ulk1 gene disruption.
    • Table S5. ULK1-dependent reduction of erythroid hyperplasia in β-thalassemic mice treated with rapamycin.
    • Table S6. Erythroid indices of individuals with β-thalassemia who provided CD34+ cells for this study.
    • Table S7. Genotypes of individuals with β-thalassemia who provided CD34+ cells for this study.
    • References (7276)

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