Reticulocyte Maturation Research Articles

Exosomes and autophagy: rekindling the vesicular waste hypothesis.

Exosomes were first described as waste carriers implicated in reticulocyte maturation but has during the past decade been associated with many other cellular functions. The biogenesis of exosomes has been extensively studied and several protein machineries have been identified to dictate their production and release. The newly discovered branches of the autophagy system implicate secretion of waste in endosomal-derived vesicles as is thought for exosome release. Many of the proteins that have been identified as responsible for the formation and release of these vesicles are the same as those identified in exosome biogenesis. In this Perspective, we discuss the possibility of exosomes being a part of the autophagy machinery and the consequences this could have on interpretation of exosome functions.

PF570 CARFILZOMIB TREATMENT CAUSES RETICULOCYTOSIS BY IMPAIRING TERMINAL ERYTHROID MATURATION IN PATIENTS WITH MULTIPLE MYELOMA

Background:The ubiquitin‐proteasome system (UPS) is crucial for cellular protein homeostasis by ensuring the degradation of redundant or misfolded proteins. In addition to its pleiotropic effects, the UPS also plays an important role in different steps of erythropoiesis. Proteasome inhibition constitutes one of the cornerstones of current multiple myeloma (MM) treatment, with three proteasome inhibitors (PI) approved for clinical use, including the reversible proteasome inhibitors (PI) bortezomib, ixazomib and irreversible PI carfilzomib. Based on a clinical observation of increased reticulocyte counts in patients with MM under treatment with carfilzomib, we hypothesized that PI can affect erythroid maturation.Aims:To address the effects of PIs on erythroid differentiation and maturation.Methods:We first retrospectively assessed the effect of carfilzomib treatment on the red cell parameters in 40 patients treated with a carfilzomib‐based regimen (carfilzomib‐dexamethasone (Kd) or carfilzomib‐lenalidomide‐dexamethasone (KRd)). Data were compared to matched cohorts of patients treated with bortezomib and the immunomodulatory drug (IMiD) lenalidomide. To address the effect of PI on erythroid differentiation in vitro, we differentiated CD34+ hematopoietic progenitor cells (HPC) into erythroid cells as previously described by the Douay group (Nature Biotechnology, 2002) in the presence of carfilzomib. To evaluate the effect of PI on terminal erythroid maturation, we purified reticulocytes from the peripheral blood of healthy volunteers, and performed ex vivo maturation in the presence the of PIs carfilzomib, bortezomib, ixazomib and MG‐132. Finally, we performed a quantitative proteomics analysis using liquid chromotrography‐mass spectrometry (LC‐MS/MS) on maturing reticulocytes to identify proteins involved in erythroid maturation.Results:We observed a specific and highly significant increase in the reticulocyte count (p < 0.001) during treatment with carfilzomib‐based regimens in patients with relapsed MM, an observation not made in a matched cohort of bortezomib‐ or lenalidomide‐treated patients. Surprisingly, this reticulocytosis was neither associated with improved hemoglobin levels nor with hemolysis. Moreover, carfilzomib treatment was not associated with increased EPO levels. Subsequent in vitro experiments demonstrated that carfilzomib did not alter the proportion of mature erythroid cells that were produced in vitro during differentiation experiments starting from CD34+ HPCs. In contrast, both continuous and pulse exposure to carfilzomib significantly impaired the terminal maturation of purified reticulocytes toward mature erythrocytes (p < 0.01). A similar effect could only be observed with reversible PI during continuous, but not during short‐term exposure. Subsequent quantitative proteomics analysis identified the RNA‐stabilizing protein PABPC1 as a candidate regulator of mRNA stabilization in terminal erythroid maturation.Summary/Conclusion:Our results indicate that proteasome inhibition with carfilzomib causes a delay of terminal erythroid maturation, which might be explained by increased mRNA stabilization through PABPC1 upregulation. This not only demonstrates the first pharmacologically induced delay in erythroid maturation but also provides new insights on physiological erythroid maturation. Moreover, delayed reticulocyte maturation should be considered as a differential diagnosis, aside from hemolytic anemia, in MM patients treated with carfilzomib.

Miro2 supplies a platform for Parkin translocation to damaged mitochondria

PINK1/Parkin-mediated mitophagy is an important process in selective removal of damaged mitochondria, in which translocation of Parkin to damaged mitochondria is recognized as an initiation step. At present, how the damaged mitochondria are selectively recognized and targeted by Parkin is not fully understood. Here we show that Miro2, an outer mitochondrial membrane protein, undergoes demultimerization from a tetramer to a monomer and alteration in mitochondrial localization upon CCCP treatment, suggesting a CCCP-induced realignment of Miro2. The realignment of Miro2 is tightly regulated by PINK1-mediated phosphorylation at Ser325/Ser430 and by Ca2+ binding to EF2 domain, which are both essential for the subsequent Parkin translocation. Interestingly, ablation of Miro2 in mouse causes delayed reticulocyte maturation, lactic acidosis and cardiac disorders. Furthermore, several Miro2 mutations found in the congenital lactic acidosis patients also disable its realignment and Parkin translocation. These findings reveal an important role of Miro2 to mediate Parkin translocation by sensing both depolarization and Ca2+ release from damaged mitochondria to ensure the accuracy of mitophagy.

Deubiquitylase USP7 regulates human terminal erythroid differentiation by stabilizing GATA1.

Ubiquitination is an enzymatic post-translational modification that affects protein fate. The ubiquitin-proteasome system (UPS) was first discovered in reticulocytes where it plays important roles in reticulocyte maturation. Recent studies have revealed that ubiquitination is a dynamic and reversible process and that deubiquitylases are capable of removing ubiquitin from their protein substrates. Given the fact that the UPS is highly active in reticulocytes, it is speculated that deubiquitylases may play important roles in erythropoiesis. Yet, the role of deubiquitylases in erythropoiesis remains largely unexplored. In the present study, we found that the expression of deubiquitylase USP7 is significantly increased during human terminal erythroid differentiation. We further showed that interfering with USP7 function, either by short hairpin RNA-mediated knockdown or USP7-specific inhibitors, impaired human terminal erythroid differentiation due to decreased GATA1 level and that restoration of GATA1 levels rescued the differentiation defect. Mechanistically, USP7 deficiency led to a decreased GATA1 protein level that could be reversed by proteasome inhibitors. Furthermore, USP7 interacts directly with GATA1 and catalyzes the removal of K48-linked poly ubiquitylation chains conjugated onto GATA1, thereby stabilizing GATA1 protein. Collectively, our findings have identified an important role of a deubiquitylase in human terminal erythroid differentiation by stabilizing GATA1, the master regulator of erythropoiesis.

T4 Mechanistic Insights on Late Stage Erythropoiesis in Burn Patients Treated with Propranolol

Burn patients suffer from Epo resistant anemia in which the Epo-independent stages of early commitment and late maturation of erythroblasts are defective. We have previously shown in mice and patients, that propranolol (prop) treatment after burn injury restored erythroid committed progenitors, while terminal maturation was still impaired. During terminal erythropoiesis, hemoglobinization is aided by alpha hemoglobin stabilizing protein (AHSP), which is an erythroblast intrinsic functional protein predominantly expressed in nucleated erythroblasts. Effective hemoglobinization results in maturation of polychromatic erythroblasts (PolyE) to reticulocytes. Here we demonstrate in PBMC derived ex vivo erythroblast cultures, AHSP is positively associated with late stage maturation in the presence of allogeneic control plasma (CP); and more so in combination with prop than with standard burn care (SBC) in burn patients. After IRB approval, blood samples were collected from 6 burn patients (age=39±16 yeas; %TBSA=32±7) at three time points on post burn days 2–4, 7–10 and 27–33. Control plasma was obtained from healthy volunteers. Fifteen PBMC samples were placed in growth factor cocktail (SCF, GMCSF, IL-3, Cyclosporine) for 0–5 days to nurture erythroid progenitors. On day 5, each sample was split into two and supplemented with either autologous burn plasma (BP) or CP along with SCF, EPO, and holotransferrin from days 5–15. We harvested cells on culture days 9, 11 and 15; subjected them to FACS and AMNIS to determine the expression (mean fluorescence intensity) and percentage of AHSP+ erythroblasts on days 9 and 11 and the percentage of Reticulocytes (Sytox- CD235a+ CD71+/- Syto16-) and PolyE (Sytox- CD235a+ CD71+/- Syto16+ SSChigh) on day 15. See figure. Overall, CP supplementation in PBMC cultures produced more reticulocytes with a proportional decrease in PolyE, indicating enhanced late stage maturation. Nonetheless, the decrease in PolyE and increase in reticulocytes are more striking in prop treated patients compared to SBC. This is consistent with about 20% increase in AHSP+ cells and AHSP expression in developing erythroblasts in prop compared to SBC. Together, CP imparts a positive influence on reticulocyte maturation by augmenting AHSP expression in PBMC derived erythroblasts. Plasma resuscitation in addition to prop treatment is a promising alternate strategy to reduce transfusions in burn patients.

A Dynamic Motility Mechanism for Reticulocyte Enucleation and Maturation

Terminal erythropoiesis involves differentiation of erythroblasts to enucleated reticulocytes, which further remove major organelles to become mature red blood cells. Several pieces of evidence indicate that the extrusion of the nucleus out of the orthochromatic erythroblasts is a fairly rapid process that lasts approximately 10 minutes. However, the detachment of the extruded nucleus from the nascent reticulocyte takes longer time, and possibly requires the aid of phagocyte to anchor the nucleus. The mechanism involved in this process is poorly understood.Previous studies have demonstrated that mDia2 is critical for erythroblast enucleation. Loss of mDia2 in mice lead to compromised cytokinesis in orthochromatic erythroblasts and decrease in enucleation. To further reveal the mechanism of the defect in enucleation in mDia2 deficient erythroid cells, we first monitored the enucleating erythroblasts using real-time microscopy. The wild type enucleating mouse erythroblast showed a dynamic motility to detach the nucleus, which can be facilitated by the macrophages. The mDia2 knockout erythroblasts retained the capacity to extrude the nucleus, even in the bi-nucleated cells. However, the nascent reticulocytes were rigid and lacked the dynamic movement to expel the nuclei. Since mDia2 is critical for linear actin polymerization, we next performed a Western blot assay of RBC ghost and found that actin and many other critical RBC cytoskeleton proteins were significantly decreased in mDia2 knockout mice (Fig 1A). Transmission electron microscope (TEM) analysis of RBC cytoskeleton revealed that spectrin filament connecting two junctional complexes was longer in mDia2 knockout RBCs (Fig 1B), which explains their rigidity and inefficient movement to detach the extruded nuclei. Further studies using stochastic optical reconstruction microscopy (STORM) revealed disorganized distribution of the major components of erythrocyte cytoskeleton, including actin (Fig 1C) and β-spectrin, in mDia2 knockout reticulocytes. The dynamic motility of the nascent reticulocytes is also required for the remodeling and removal of proteins to further mature into RBCs. We found an increase in proteins such as CD71 and CD44, as well as DNA/RNAs, in mDia2 knockout reticulocytes. Mechanistic studies revealed that mDia2 functions on vesicle trafficking through its interaction with Chmp5, one of the critical components of the ESCRT-III complex involved in vesicle formation and trafficking. This interaction stabilized Chmp5 since its level was dramatically reduced in mDia2 knockout erythroblasts and reticulocytes (Fig 1D-E).Our study reveals a critical role of the dynamic motility of the nascent reticulocyte in enucleation and maturation. Abnormal membrane cytoskeleton in mDia2 null mice compromises these processes. In one hand, mDia2 mediated actin polymerization ensures the flexibility of the erythrocyte cytoskeleton. On the other hand, mDia2 also involves vesicle biogenesis and discharging through the regulation of ESCRT complexes during reticulocyte maturation. Our study thus sheds lights on the understanding of reticulocyte maturation mechanisms. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Absolute proteome quantification of highly purified populations of circulating reticulocytes and mature erythrocytes

AbstractReticulocytes produced in the bone marrow undergo maturation in the bloodstream to give rise to erythrocytes. Although the proteome of circulating red cells has been the subject of several reports, the cellular populations used for these studies were never completely devoid of reticulocytes. In our current study, we used highly purified erythrocyte and reticulocyte populations to quantify the absolute expression levels of the proteins in each cell population. Erythrocytes and reticulocytes were purified in a multistep process involving cellulose chromatography, Percoll gradient centrifugation, and fluorescence cell sorting after thiazole orange labeling. Proteins were analyzed by mass spectrometry from whole cells and erythrocyte plasma membrane (ghosts), leading to the identification and quantification of 2077 proteins, including 654 that were reticulocyte-specific. Absolute quantifications of these proteins were made using the mean corpuscular hemoglobin content of the cells as a standard. For each protein, we calculated the percentage loss during the terminal stages of reticulocyte maturation and the percentage of association with the plasma membrane. In addition, we used modified adenosine triphosphate and adenosine diphosphate molecules that enable the transfer of a biotin molecule to the catalytic sites of kinases to isolate active kinases in the erythrocytes and determined the absolute expression of 75 protein kinases and the modification of their expression during reticulocyte maturation. Our findings represent the first absolute quantification of proteins that are specifically expressed in normal erythrocytes with no detectable contamination by reticulocytes. Our findings thus represent a reference database for the future proteomic analysis of pathological erythrocytes.

Proteomics study of human cord blood reticulocyte-derived exosomes

Reticulocyte-derived exosomes (Rex), extracellular vesicles of endocytic origin, were initially discovered as a cargo-disposal mechanism of obsolete proteins in the maturation of reticulocytes into erythrocytes. In this work, we present the first mass spectrometry-based proteomics of human Rex (HuRex). HuRex were isolated from cultures of human reticulocyte-enriched cord blood using different culture conditions and exosome isolation methods. The newly described proteome consists of 367 proteins, most of them related to exosomes as revealed by gene ontology over-representation analysis and include multiple transporters as well as proteins involved in exosome biogenesis and erythrocytic disorders. Immunoelectron microscopy validated the presence of the transferrin receptor. Moreover, functional assays demonstrated active capture of HuRex by mature dendritic cells. As only seven proteins have been previously associated with HuRex, this resource will facilitate studies on the role of human reticulocyte-derived exosomes in normal and pathological conditions affecting erythropoiesis.

Equine blood reticulocytes: reference intervals, physiological and pathological changes

High-sensitivity haematology analysers detect equine blood reticulocytes, but their analysis and quantitation is largely unstudied. We assessed whether equine reticulocytes are affected by pathological or physiological factors, determined reference intervals, and examined reticulocyte and related, erythron parameters using 6-years’ data (Advia 2120) from our hospital archives. Data was categorised according to horse age (foals < 12 months), breed (hot-, warm-, cold-blooded), presence of anaemia and various diseases. In adults, reticulocytes (× 109/L) were 20% lower in cold-blooded horses compared to others (4.14 ± 2.3, 25 vs 5.15 ± 3.3, 157; data shown as mean ± SD, n; p < 0.05.) There was no gender effect. In thoroughbreds, foals had 50% higher reticulocytes than adults (7.5 ± 4.5, 18 vs 5.0 ± 3.5, 56). We determined the first reference interval for healthy adult horses as 5.0 ± 3.2,182, 1.16–13.1. Reticulocyte counts were 120% higher with marked anaemia (11.0 ± 9.4, 6) but 36% with other diseases (6.9 ± 4.2, 74), versus reference mean. Erythrocyte size (MCV)/reticulocyte haemoglobin content (CHr) were 21/14% lower in foals versus adults and 12/37% higher with marked anaemia. Immature reticulocytes (IRF-H) were 60% lower in foals versus adult thoroughbreds, and 87% with marked anaemia. In conclusion, equine reticulocytes are only mildly increased by physiological parameters (breed, age, but not gender), various non-blood diseases (colic, dysproteinaemia) and marked anaemia. Reticulocyte counts are substantially lower in horses versus other species; their clinical use is limited to marked anaemias. This is consistent with the greater erythrocyte storage and buffering capacity of the spleen of the horse. Finally, this study supports the theory of a reticulocyte maturation role for equine spleen.

Non-muscle myosin II drives vesicle loss during human reticulocyte maturation.

The process of maturation of reticulocytes into fully mature erythrocytes that occurs in the circulation is known to be characterized by a complex interplay between loss of cell surface area and volume, removal of remnant cell organelles and redundant proteins, and highly selective membrane and cytoskeletal remodeling. However, the mechanisms that underlie and drive these maturational processes in vivo are currently poorly understood and, at present, reticulocytes derived through in vitro culture fail to undergo the final transition to erythrocytes. Here, we used high-throughput proteomic methods to highlight differences between erythrocytes, cultured reticulocytes and endogenous reticulocytes. We identify a cytoskeletal protein, non-muscle myosin IIA (NMIIA) whose abundance and phosphorylation status differs between reticulocytes and erythrocytes and localized it in the proximity of autophagosomal vesicles. An ex vivo circulation system was developed to simulate the mechanical shear component of circulation and demonstrated that mechanical stimulus is necessary, but insufficient for reticulocyte maturation. Using this system in concurrence with non-muscle myosin II inhibition, we demonstrate the involvement of non-muscle myosin IIA in reticulocyte remodeling and propose a previously undescribed mechanism of shear stress-responsive vesicle clearance that is crucial for reticulocyte maturation.

Uncoupling of CD71 shedding with mitochondrial clearance in reticulocytes in a subset of myelodysplastic syndromes.

Reticulocytes shed CD71 from the cell membrane and eliminate mitochondria during terminal maturation, but it is unknown whether these two events are coordinated. We demonstrate that timely removal of CD71 is coupled with mitochondrial clearance, which can be disrupted by null mutation of immediate early response gene X-1 (IEX-1), leading to generation of aberrant CD71-positive and mitochondria-negative (CD71+Mito-) reticulocytes. CD71+Mito- reticulocytes were also present in a subset of patients with myelodysplastic syndromes (MDS) in direct proportion to reduced mitochondrial membrane potential (∆ψm). Mitochondrial abnormality caused by either IEX-1 deficiency or agents that dissipate ∆ψm could trigger premature clearance of mitochondria in reticulocytes. Premature clearance of mitochondria or addition of anti-oxidants lowered intracellular reactive oxygen species (ROS) that in turn hindered CD71 shedding and reticulocyte maturation. In contrast, introduction of ROS accelerated CD71 shedding via release of exosomes that contained a high proportion of Fe3+ over Fe2+, suggesting dual functions of CD71 shedding both in removal of toxic Fe3+ from reticulocytes and in limiting importation of Fe3+ into the cells. These observations emphasize the coordination of mitochondrial and CD71 clearance in erythroid terminal maturation and offer new insights into a role for mitochondrial degeneration in the pathogenesis of some MDS-associated anemia.

The Shape Shifting Story of Reticulocyte Maturation

The final steps of erythropoiesis involve unique cellular processes including enucleation and reorganization of membrane proteins and the cytoskeleton to produce biconcave erythrocytes. Surprisingly this process is still poorly understood. In vitro erythropoiesis protocols currently produce reticulocytes rather than biconcave erythrocytes. In addition, immortalized lines and iPSC-derived erythroid cell suffer from low enucleation and suboptimal final maturation potential. In light of the increasing prospect to use in vitro produced erythrocytes as (personalized) transfusion products or as therapeutic delivery agents, the mechanisms driving this last step of erythropoiesis are in dire need of resolving. Here we review the elusive last steps of reticulocyte maturation with an emphasis on protein sorting during the defining steps of reticulocyte formation during enucleation and maturation.

In-depth phenotypic characterization of reticulocyte maturation using mass cytometry

Progress towards an in-depth understanding of the final steps of the erythroid lineage development is paramount for many hematological diseases. We have characterized the final stages of reticulocyte maturation from bone marrow to peripheral blood using for the first time single-cell Mass Cytometry (CyTOF). We were able to measure the expression of 31 surface markers within a single red blood cell (RBC). We demonstrate the validity of CyTOF for RBC phenotyping by confirming the progressive reduction of transferrin receptor 1 (CD71) during reticulocyte maturation to mature RBC. We highlight the high-dimensional nature of mass cytometry data by correlating the expression of multiple proteins on individual RBCs. We further describe a more drastic reduction pattern for a component of the alpha4/beta1 integrin CD49d at the very early steps of reticulocyte maturation in bone marrow and directly linked with the mitochondria remnants clearance pattern. The enhanced and accurate RBC phenotyping potential of CyTOF described herein could be beneficial to decipher RBC preferences, as well as still not well understood receptor-ligand interaction of some hemotropic parasites such as the malaria causing agent Plasmodium vivax.

Continuous Change in Membrane and Membrane-Skeleton Organization During Development From Proerythroblast to Senescent Red Blood Cell.

Within the context of erythropoiesis and the possibility of producing artificial red blood cells (RBCs) in vitro, a most critical step is the final differentiation of enucleated erythroblasts, or reticulocytes, to a fully mature biconcave discocyte, the RBC. Reviewed here is the current knowledge about this fundamental maturational process. By combining literature data with our own experimental evidence we propose that the early phase in the maturation of reticulocytes to RBCs is driven by a membrane raft-based mechanism for the sorting of disposable membrane proteins, mostly the no longer needed transferrin receptor (TfR), to the multivesicular endosome (MVE) as cargo of intraluminal vesicles that are subsequently exocytosed as exosomes, consistently with the seminal and original observation of Johnstone and collaborators of more than 30 years ago (Pan BT, Johnstone RM. Cell. 1983;33:967-978). According to a strikingly selective sorting process, the TfR becomes cargo destined to exocytosis while other molecules, including the most abundant RBC transmembrane protein, band 3, are completely retained in the cell membrane. It is also proposed that while this process could be operating in the early maturational steps in the bone marrow, additional mechanism(s) must be at play for the final removal of the excess reticulocyte membrane that is observed to occur in the circulation. This processing will most likely require the intervention of the spleen, whose function is also necessary for the continuous remodeling of the RBC membrane all along this cell's circulatory life.

Secretory factors from OP9 stromal cells delay differentiation and increase the expansion potential of adult erythroid cells in vitro

Development of in vitro culture systems for the generation of red blood cells is a goal of scientists globally with the aim of producing clinical grade products for transfusion. Although mature reticulocytes can be efficiently generated by such systems, the numbers produced fall short of that required for therapeutics, due to limited proliferative capacity of the erythroblasts. To overcome this hurdle, approaches are required to increase the expansion potential of such culture systems. The OP9 mouse stromal cell line is known to promote haematopoietic differentiation of pluripotent stem cells, however an effect of OP9 cells on erythropoiesis has not been explored. In this study, we show not only OP9 co-culture, but factors secreted by OP9 cells in isolation increase the proliferative potential of adult erythroid cells by delaying differentiation and hence maintaining self-renewing cells for an extended duration. The number of reticulocytes obtained was increased by approximately 3.5-fold, bringing it closer to that required for a therapeutic product. To identify the factors responsible, we analysed the OP9 cell secretome using comparative proteomics, identifying 18 candidate proteins. These data reveal the potential to increase erythroid cell numbers from in vitro culture systems without the need for genetic manipulation or co-culture.

VPS4A : A Novel Candidate Gene for Congenital Dyserythropoietic Anemia

CDAR (ClinicalTrials.gov Identifier: NCT02964494), a registry for patients with Congenital Dyserythropoietic Anemia (CDA) in North America, was recently created with the goal to provide a longitudinal database and associated biorepository to facilitate natural history studies and research on the molecular pathways involved in the pathogenesis of CDAs. A 2 y.o. female patient with transfusion dependent anemia, pathologic diagnosis of Congenital Dyserythropoietic Anemia type I (CDA-I), and neurodevelopmental delay was enrolled in CDAR. Next Generation sequencing and deletion/duplication assay identified no mutations in the known CDA-associated genes, including CDAN1 and C15orf41, which are causative for CDA-I. Whole-exome sequencing for the patient and her parents (family-trio design) revealed a novel, de novo VPS4A missense variant located in the last codon of exon 8, potentially affecting splicing. VPS4A is an ATPase which, in association with the endosomal sorting complex required for transport (ESCRT), has been shown to play a critical role in cell division of HeLa cells in vitro, concentrating at the spindle poles during mitosis and at the midbody during cytokinesis. The aim of this work is to validate the pathogenetic role of the VPS4A variant for CDA and further investigate the role of VPS4A in erythroblast mitosis and cytokinesis.Central review of the patient's bone marrow aspirate smears revealed bi-nucleated erythroblasts in the range of 3-7%, a criterion compatible with CDA-I. However, cytoplasmic bridges were noted (arrows in Figure 1A) rather than the nuclear bridges typical of CDA-I. Immunofluorescence staining performed on erythroblasts generated ex vivo from normal CD34+ cells verified that VPS4A localizes to the spindle poles during mitosis and the midbody during cytokinesis in dividing human erythroid cells analyzed by Imaging Flow Cytometry (Figure 1B). The level of VPS4A mRNA expression in the patient's reticulocytes was evaluated by qPCR using three different sets of primers and found to be decreased by 55-70% compared to control reticulocytes and knock-down of VPS4A in normal CD34+ cells resulted in erythroid cultures enriched in binucleated cells. Induced pluripotent stem cells (iPSCs) were generated from the patient's peripheral blood mononuclear cells after the family's consent. Erythroblasts produced from these iPSCs exhibited decreased VPS4A localization at the spindle poles and midbody and fail to divide properly, frequently maintaining cytoplasmic bridges as seen in the patient's bone marrow. Additionally, flow cytometry analysis of the patient's peripheral blood cells stained with anti-CD71 for the transferrin receptor and Thiazol Orange (TO) for RNA revealed a unique cell population which is TO negative, yet CD71 positive implying that VPS4A is also involved in reticulocyte maturation, likely participating in vesicle formation and the normal exocytosis of the transferrin receptor.VPS4A appears to play a critical role in erythroblast mitosis and cytokinesis, as well as erythrocyte maturation, and is a novel candidate gene for congenital dyserythropoietic anemia.Figure 1. A) Binucleated erythroblasts and cytoplasmic bridges (arrows) were noted on the patient's bone marrow aspirate smears. B) VPS4A localizes at the spindle poles (upper image) and midbody (lower image) in normal human erythroblasts. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Fyn Kinase Is Involved in EPO Receptor Signaling and Is Required to Harmonize the Response to Oxidation

Erythropoiesis is a complex multistep process during which committed erythroid progenitors undergo terminal differentiation to produce circulating mature red cells. Erythroid differentiation is characterized by the production of reactive oxygen species (ROS) both in response to erythropoietin (EPO) and to the large amount of iron imported into the cells for heme biosynthesis. During erythropoiesis, ROS might function as second messenger by modulating intracellular signaling pathways. Fyn, an Src kinase, has been previously reported to participate in signaling pathways in response to ROS in various cell types. Here, we explore the potential contribution of Fyn to normal and stress erythropoiesis by studying 2-4 months-old Fyn knockout mouse strain (Fyn-/-) and C57B6/2J as wild-type controls. Fyn-/- mice showed a mild compensated microcytic anemia associated with signs of dyserythropoiesis. Increased ROS levels and Annexin-V+ cells were presented in all Fyn-/- erythroblast subpopulations compared to wild-type, suggesting a possible reduction in the efficiency of erythropoietin (EPO) signaling pathway in the absence of Fyn. Indeed, in Fyn-/- erythroblasts we observed a reduction in Tyr-phosphorylation state of EPO-R associated with a compensatory activation of Jak2 without major change in Lyn activity. A reduction in STAT5 activation resulting in down-regulation of Cish, a known direct STAT5 target gene, was noted in Fyn-/- erythroblasts. This was paralleled by a reduction in GATA1 and increased HSP70 nuclear translocation compared to wild type, supporting a higher cellular pro-oxidant environment in the absence of Fyn. Using the vitro cell forming colony unit assay, we found a lower in CFU-E and BFU-E cells production, which once again was associated with decreased activation of EPO mediated cascade in the absence of Fyn. To explore the possible role of Fyn in stress erythropoiesis, mice were treated with either phenylhydrazine (PHZ) or doxorubicin (Doxo). Fyn-/- mice showed prolonged anemia after either PHZ or Doxo treatment with a delayed hematologic recovery compared to wild-type animals. When we analyzed the expression of a battery of ARE-genes related to oxidative response such as catalase, Gpx, heme-oxygenase 1 and peroxiredoxin-2, we noted up-regulated expression of these genes in sorted Fyn-/- erythroblasts compared to wild-type cells. In agreement, we observed increased activation of the redox-sensitive transcriptional factor Nrf2 targeting ARE-genes, whose regulation has been previously linked to Fyn. In fact, Nrf2 is switched-off by Fyn, ubiquitylated and delivered to the autophagosome by the p62 cargo protein. In Fyn-/- sorted erythroblasts, we observed (i) accumulation of p62 in large clusters; and (ii) reduction of Nrf2-p62 complex compared to wild-type cells. To address the question whether the perturbation of Nrf2-p62 system results in impairment of autophagy in the absence of Fyn, we used Lysotrack to explore late phases of autophagy. Lysosomal progression was defective in Fyn-/- reticulocytes and it was associated with accumulation of p62 during in vitro reticulocyte maturation. These data indicate that the absence of Fyn blocks the Nrf2 post-induction response to oxidation, resulting in impaired autophagy. To validate our working hypothesis, we treated Fyn-/- mice with Rapamycin, an inducer of autophagy. In Fyn-/- mice, Rapamycin treatment resulted in decrease dyserythropoiesis, ROS levels and Annexin V+ cells, associated with reduction in accumulation of p62 in Fyn-/- erythroblasts. As a proof of concept, we treated both mouse strains with PHZ with or without Rapamycin. This latter worsened PHZ induced acute anemia in wild-type mice but not in Fyn-/- animals. Collectively, our data enabled us to document a novel role for Fyn in erythropoiesis, contributing to EPO-R activation and harmonizing the Nrf2-p62 adaptive cellular response against oxidation during normal and more importantly in stress erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

Quantitative mass spectrometry of human reticulocytes reveal proteome‐wide modifications during maturation

Erythropoiesis is marked by progressive changes in morphological, biochemical and mechanical properties of erythroid precursors to generate red blood cells (RBC). The earliest enucleated forms derived in this process, known as reticulocytes, are multi-lobular and spherical. As reticulocytes mature, they undergo a series of dynamic cytoskeletal re-arrangements and the expulsion of residual organelles, resulting in highly deformable biconcave RBCs (normocytes). To understand the significant, yet neglected proteome-wide changes associated with reticulocyte maturation, we undertook a quantitative proteomics approach. Immature reticulocytes (marked by the presence of surface transferrin receptor, CD71) and mature RBCs (devoid of CD71) were isolated from human cord blood using a magnetic separation procedure. After sub-fractionation into triton-extracted membrane proteins and luminal samples (isobaric tags for relative and absolute quantitation), quantitative mass spectrometry was conducted to identify more than 1800 proteins with good confidence and coverage. While most structural proteins (such as Spectrins, Ankyrin and Band 3) as well as surface glycoproteins were conserved, proteins associated with microtubule structures, such as Talin-1/2 and ß-Tubulin, were detected only in immature reticulocytes. Atomic force microscopy (AFM)-based imaging revealed an extended network of spectrin filaments in reticulocytes (with an average length of 48nm), which shortened during reticulocyte maturation (average spectrin length of 41nm in normocytes). The extended nature of cytoskeletal network may partly account for increased deformability and shape changes, as reticulocytes transform to normocytes.

DARC extracellular domain remodeling in maturating reticulocytes explains Plasmodium vivax tropism

AbstractPlasmodium vivax is the most prevalent parasite species that causes malaria in humans and exclusively infects reticulocytes. Reticulocyte infection is facilitated by P vivax Duffy binding protein (DBP), which utilizes DARC (Duffy antigen receptor for chemokines) as an entry point. However, the selective tropism of P vivax for transferrin receptor (CD71)-positive reticulocytes remained unexplained, given the constitutive expression of DARC during reticulocyte maturation. CD71/RNA double staining of reticulocytes enriched from adult peripheral blood reveals 4 distinct reticulocyte populations: CD71high/RNAhigh (∼0.016%), CD71low/RNAhigh (∼0.059%), CD71neg/RNAhigh (∼0.37%), CD71neg/RNAlow (∼0.55%), and erythrocytes CD71neg/RNAneg (∼99%). We hypothesized that selective association of DBP with a small population of immature reticulocytes could explain the preference of P vivax for reticulocytes. Binding of specific monoclonal anti-DARC antibodies and recombinant DBP to CD71high/RNAhigh reticulocytes was significantly higher compared with other reticulocyte populations and erythrocytes. Interestingly, the total DARC protein throughout reticulocyte maturation was constant. The data suggest that selective exposure of the DBP binding site within DARC is key to the preferential binding of DBP to immature reticulocytes, which is the potential mechanism underlying the preferential infection of a reticulocyte subset by P vivax.

BNIP3L/NIX-mediated mitophagy protects against ischemic brain injury independent of PARK2

ABSTRACTCerebral ischemia induces massive mitochondrial damage. These damaged mitochondria are cleared, thus attenuating brain injury, by mitophagy. Here, we identified the involvement of BNIP3L/NIX in cerebral ischemia-reperfusion (I-R)-induced mitophagy. Bnip3l knockout (bnip3l−/−) impaired mitophagy and aggravated cerebral I-R injury in mice, which can be rescued by BNIP3L overexpression. The rescuing effects of BNIP3L overexpression can be observed in park2−/− mice, which showed mitophagy deficiency after I-R. Interestingly, bnip3l and park2 double-knockout mice showed a synergistic mitophagy deficiency with I-R treatment, which further highlighted the roles of BNIP3L-mediated mitophagy as being independent from PARK2. Further experiments indicated that phosphorylation of BNIP3L serine 81 is critical for BNIP3L-mediated mitophagy. Nonphosphorylatable mutant BNIP3LS81A failed to counteract both mitophagy impairment and neuroprotective effects in bnip3l−/− mice. Our findings offer insights into mitochondrial quality control in ischemic stroke and bring forth the concept that BNIP3L could be a potential therapeutic target for ischemic stroke, beyond its accepted role in reticulocyte maturation.