In β-thalassemia, accumulated free α-globin forms intracellular precipitates that impair erythroid cell maturation and viability. Protein quality control systems mitigate β-thalassemia pathophysiology by degrading toxic free α-globin, although the associated mechanisms are poorly understood. We show that loss of the autophagy-activating Unc-51–like kinase 1 (Ulk1) gene in β-thalassemic mice reduces autophagic clearance of α-globin in red blood cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy-related 5 (Atg5) gene has relatively minor effects. Systemic treatment with the mTORC1 inhibitor rapamycin reduces α-globin precipitates and lessens pathologies in β-thalassemic mice via an ULK1-dependent pathway. Similarly, rapamycin reduces free α-globin accumulation in erythroblasts derived from CD34+cells of β-thalassemic individuals. Our findings define a drug-regulatable pathway for ameliorating β-thalassemia.

BackgroundPatients with heterozygous germline mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) experience autoimmunity and lymphoid hyperplasia.ObjectivesBecause regulation of the phosphoinositide 3-kinase (PI3K) pathway is critical for maintaining regulatory T (Treg) cell functions, we investigate Treg cells in patients with heterozygous germline PTEN mutations (PTEN hamartoma tumor syndrome [PHTS]).MethodsPatients with PHTS were assessed for immunologic conditions, lymphocyte subsets, forkhead box P3 (FOXP3)+ Treg cell levels, and phenotype. To determine the functional importance of phosphatases that control the PI3K pathway, we assessed Treg cell induction in vitro, mitochondrial depolarization, and recruitment of PTEN to the immunologic synapse.ResultsAutoimmunity and peripheral lymphoid hyperplasia were found in 43% of 79 patients with PHTS. Immune dysregulation in patients with PHTS included lymphopenia, CD4+ T-cell reduction, and changes in T- and B-cell subsets. Although total CD4+FOXP3+ Treg cell numbers are reduced, frequencies are maintained in the blood and intestine. Despite pathogenic PTEN mutations, the FOXP3+ T cells are phenotypically normal. We show that the phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP) downstream of PTEN is highly expressed in normal human Treg cells and provides complementary phosphatase activity. PHLPP is indispensable for the differentiation of induced Treg cells in vitro and Treg cell mitochondrial fitness. PTEN and PHLPP form a phosphatase network that is polarized at the immunologic synapse.ConclusionHeterozygous loss of function of PTEN in human subjects has a significant effect on T- and B-cell immunity. Assembly of the PTEN-PHLPP phosphatase network allows coordinated phosphatase activities at the site of T-cell receptor activation, which is important for limiting PI3K hyperactivation in Treg cells despite PTEN haploinsufficiency.

No abstract

Most cells can eliminate unstable or misfolded proteins through quality control mechanisms. In the inherited red blood cell disorder β-thalassemia, mutations in the β-globin gene (HBB) lead to a reduction of the corresponding protein and the accumulation of cytotoxic free α-globin, which causes maturation arrest and apoptosis of erythroid precursors and reductions in the lifespan of circulating red blood cells. We showed previously that excess α-globin is eliminated by ULK1-dependent autophagy and that stimulation of this pathway by systemic mTORC1 inhibition alleviates β-thalassemia pathologies. We show here that disruption of the bi-cistronic microRNA locus miR-144/451 alleviates β-thalassemia by reducing mTORC1 activity and stimulating ULK1-mediated autophagy of free α-globin through two mechanisms. Loss of miR-451 upregulated its target mRNA, Cab39, which encodes a cofactor for LKB1, a serine-threonine kinase that phosphorylates and activates the central metabolic sensor, AMPK. The resultant enhancement of LKB1 activity stimulated AMPK and its downstream effects, including repression of mTORC1 and direct activation of ULK1. Additionally, loss of miR-144/451 inhibited the expression of erythroblast transferrin receptor 1 (TfR1), causing intracellular iron restriction, which has been shown to inhibit mTORC1, reduce free α-globin precipitates and improve hematological indices in β-thalassemia. The beneficial effects of miR-144/451 loss in β-thalassemia were inhibited by disruption of the Cab39 or Ulk1 genes. Our findings link the severity of a common hemoglobinopathy to a highly expressed erythroid microRNA locus and to a fundamental, metabolically regulated protein quality control pathway that is amenable to therapeutic manipulation.

Erythroid maturation is coordinated to maximize the production of hemoglobin A heterotetramers (α2β2) and minimize the accumulation of potentially toxic free α-or βglobin subunits. In β-thalassemia, mutations in the β-globin gene cause a build-up of free α-globin, which forms intracellular precipitates that impair erythroid cell maturation and 5 viability. Protein quality-control systems mitigate β-thalassemia pathophysiology by degrading toxic free α-globin. We show that loss of the Unc 51-like autophagy-activating kinase gene Ulk1 in β-thalassemic mice reduces autophagic clearance of α-globin in red cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy gene Atg5 has minimal effects. Systemic treatment with rapamycin 10 to inhibit the ULK1 inhibitor mTORC1 reduces α-globin precipitates and lessens pathologies in β-thalassemic mice, but not in those lacking Ulk1. Similarly, rapamycin reduces free α-globin accumulation in erythroblasts derived from β-thalassemic patient CD34 + hematopoietic progenitors. Our findings identify a new, drug-regulatable pathway for ameliorating β-thalassemia. 15 One Sentence Summary:Rapamycin alleviates -thalassemia by stimulating ULK1-dependent autophagy of toxic free -globin. 20