Florent M. Martin

SOD2 Deficient Erythroid Cells Up-Regulate Transferrin Receptor and Down-Regulate Mitochondrial Biogenesis and Metabolism

Background Mice irradiated and reconstituted with hematopoietic cells lacking manganese superoxide dismutase (SOD2) show a persistent hemolytic anemia similar to human sideroblastic anemia (SA), including characteristic intra-mitochondrial iron deposition. SA is primarily an acquired, clonal marrow disorder occurring in individuals over 60 years of age with uncertain etiology. Methodology/Principal Findings To define early events in the pathogenesis of this murine model of SA, we compared erythroid differentiation of Sod2-/- and normal bone marrow cells using flow cytometry and gene expression profiling of erythroblasts. The predominant transcriptional differences observed include widespread down-regulation of mitochondrial metabolic pathways and mitochondrial biogenesis. Multiple nuclear encoded subunits of complexes I-IV of the electron transport chain, ATP synthase (complex V), TCA cycle and mitochondrial ribosomal proteins were coordinately down-regulated in Sod2-/- erythroblasts. Despite iron accumulation within mitochondria, we found increased expression of transferrin receptor, Tfrc, at both the transcript and protein level in SOD2 deficient cells, suggesting deregulation of iron delivery. Interestingly, there was decreased expression of ABCb7, the gene responsible for X-linked hereditary SA with ataxia, a component required for iron-sulfur cluster biogenesis. Conclusions/Significance These results indicate that in erythroblasts, mitochondrial oxidative stress reduces expression of multiple nuclear genes encoding components of the respiratory chain, TCA cycle and mitochondrial protein synthesis. An additional target of particular relevance for SA is iron:sulfur cluster biosynthesis. By decreasing transcription of components of cluster synthesis machinery, both iron utilization and regulation of iron uptake are impacted, contributing to the sideroblastic phenotype.

The familial Parkinson's disease gene DJ-1 (PARK7) is expressed in red cells and plays a role in protection against oxidative damage.

The antioxidant enzyme manganese superoxide dismutase (SOD2) serves as the primary defense against mitochondrial superoxide. Impaired SOD2 activity in murine hematopoietic cells affects erythroid development, resulting in anemia characterized by intra-mitochondrial iron deposition, reticulocytosis and shortened red cell life span. Gene expression profiling of normal and SOD2 deficient erythroblasts identified the Parkinsons disease locus DJ-1 (Park7) as a differentially expressed transcript. To investigate the role of DJ-1 in hematopoietic cell development and protection against oxidative stress caused by Sod2 loss, we evaluated red cell parameters, reticulocyte count, red cell turnover and reactive oxygen species production in DJ-1 knockout animals and chimeric animals lacking both SOD2 and DJ-1 in hematopoietic cells generated by fetal liver transplantation. We also investigated DJ-1 protein expression in primary murine erythroid and erythroleukemia cells (MEL). Loss of DJ-1 exacerbates the phenotype of SOD2 deficiency, increasing reticulocyte count and decreasing red cell survival. Using MEL cells, we show that DJ-1 is up-regulated at the protein level during erythroid differentiation. These results indicate that DJ-1 plays a physiologic role in protection of erythroid cells from oxidant damage, a function unmasked in the context of oxidative stress.

Purification and characterization of sideroblasts from patients with acquired and hereditary sideroblastic anaemia.

The sideroblastic anemias (SAs) are a heterogeneous group of inherited (rare) and acquired (relatively common) disorders of erythroid development characterized by iron accumulation within the mitochondria of developing erythroid cells, i.e. ringed sideroblasts (RS). Mutations in a few genes cause types of hereditary SA—including the gene that encodes the haem biosynthetic enzyme 5-aminolevulinate synthase (Cotter, et al 1994), a putative mitochondrial transporter ATP binding cassette b7 (Allikmets, et al 1999) an RNA modifying enzyme, pseudouridine synthase (Bykhovskaya, et al 2004), and a mitochondrial localized protein involved in iron-sulfur cluster biogenesis, glutaredoxin 5 (Camaschella, et al 2007). A large deletion of mitochondrial DNA in Pearson marrow pancreas syndrome causes SA in the context of a multi-system mitochondrial disorder (Rotig, et al 1990). Acquired SA is most commonly seen in myelodysplastic syndromes (MDS), where standard karyotypic analyses have not defined cytogenetic abnormalities that predict the presence of ringed sideroblasts. The significance of mitochondrial DNA mutations in the pathogenesis of SA and MDS remains controversial, with conflicting data as to whether mutation frequency is increased in marrow cells from patients with this group of disorders (Gattermann 2000, Reddy, et al 2002, Shin, et al 2003), and little direct evidence to link specific mutations with disease (Wulfert, et al 2008). Herein, we describe purification of sideroblasts from human marrow samples employing a simple, magnetic column-based method that was initially developed using a mouse model system (Martin et al 2005), and present characterization of the purified human cells. This study included four males and two females, aged 70−85 years, with MDS and RS (3 refractory anemia with ringed sideroblasts [RARS], 3 multilineage dysplasia or excess blasts). One RARS patient provided two specimens for this study 10 months apart. One of the MDS patients progressed to acute myeloid leukemia ∼4 months after a sample was obtained. The seventh patient in this study was a 26-year-old male with X-linked sideroblastic anemia. All marrow samples were collected with the approval of the Scripps Research Institute human subjects committee. The control marrow specimens lacked RS or evidence of dysplasia. Sideroblasts were purified by adaptation of a simple magnetic column-based method as described previously for purification of murine siderocytes (Martin et al 2005). Reactive oxygen species (ROS) production and mitochondrial membrane potential (ΔΨm) flow cytometry (fluorescent-activated cell sorting) assays, and protein carbonyl determination (oxyblot) were performed as described previously (Martin et al 2005). Using a murine model of SA, we have previously demonstrated that the increased intracellular iron characteristic of sideroblasts or siderocytes (enucleated red cells containing iron-loaded mitochondria) can be exploited to obtain a highly purified population of these cells (Martin et al 2005). Here, we utilized the same method (passage of a cell suspension over a magnetic column in absence of any magnetic bead affinity reagent) for purification of sideroblasts and siderocytes from 8 fresh human marrow specimens. 0.29 ± 0.07% vs. 0.08 ± 0.01% of cells were recovered in the column-bound fraction (CBF) relative to starting material (SM) in diseased (N = 8) vs. normal (N = 2) whole marrows, respectively. This represents a 3.62-fold greater proportion of magnet+ cells in diseased than in normal samples (Fig. 1A). Cell counts included both nucleated cells and erythrocytes in the starting marrow specimens and purified samples. Fig.1 Magnetic purification of marrow aspirates from patients with RS allowed purification of iron-laden erythroid precursors Magnetic purification of bone marrow suspensions showed a significant enrichment in glycophorin A (GPA)+ and transferrin receptor (CD71)+ iron-overloaded erythroblasts, i.e. sideroblasts (35.07 ± 6.06% of gated cells in CBF vs. 2.27 ± 0.67 and 2.40 ± 1.35% of gated cells in SM and column flow-through (FT), respectively; N=8; ***, P 90% of purified cells were erythroid (erythroblasts to siderocytes) (Fig. 1B). Perls iron stain demonstrated significant iron accumulation within cells in the magnet-purified fraction - including nucleated RS and siderocytes. Purified sideroblasts showed a significantly increased mitochondrial membrane potential, ΔΨm, with geometric mean fluorescence intensity (GeoMFI) of 57.05 ± 11.79 relative to SM and FT fractions (5.60 ± 0.87 and 5.54 ± 0.91 GeoMFI, respectively; N=8; ***, P<0.0001; Fig. 2B). These results are similar to those observed when purifying murine sideroblasts/siderocytes in a mouse model of SA secondary to loss of the intramitochondrial antioxidant protein superoxide dismutase 2 (Martin et al 2005). Elevation of the mitochondrial membrane potential implies an increase in the H+ gradient within the mitochondria, and may reflect a defect in the distal portions of the electron transport chain or a defect in mitochondrial ATP synthesis. Fig.2 Purified sideroblasts produced high levels of ROS, showed altered ΔΨm and increased oxidative damage to proteins ROS sensitive dyes dihydroethidium (DHE—sensitive to superoxide) and 5,6 chloromethyl 2’,7’ dichlorodihydro-fluorescein diacetate (CM-H2DCFDA—a fluorescein derivative sensitive to peroxide and mixed ROS) were used to measure real-time peroxide and superoxide production in unfractionated marrow, magnet purified and flow through fractions. Most cells in the magnet-purified fraction (CBF) produced ROS, whereas few cells from the SM and FT fractions showed significant dye oxidation (71.27 ± 5.05% of gated cells vs. 1.26 ± 0.22 and 0.89 ± 0.16% of gated cells, respectively; N=8; ***, P<0.0001; Fig. 2 A). These results were consistent with comparisons of magnet purified siderocytes from the murine model system (Martin et al 2005). Sideroblasts purified from 3 patients (CBF fractions) showed a significant increase in the amount and complexity of protein oxidative modification, measured as carbonyls (Fig. 2 C), when compared with an equivalent amount of protein from starting material, or from cells that passed through the magnetic column. This is similar to the enrichment for oxidized proteins observed when purifying sideroblasts/siderocytes from murine marrow or peripheral blood (Martin et al 2005). This association between excess iron and protein oxidation raises the possibility that redox active iron is a source of protein-damaging ROS via Fenton chemistry in developing erythrocytes. We have demonstrated a simple and effective method for the enrichment of sideroblasts/siderocytes from marrow specimens of patients with SA or with myelodysplasia with ringed sideroblasts. These cells, purified on a magnet to take advantage of their high iron content, show evidence of increased oxidant production, altered mitochondrial function and oxidative damage to protein. We anticipate that this purified cell population will be useful for additional analyses to delineate other molecular and biochemical lesions characteristic of acquired SA.