ALBERT

Description: The adducins are a family of three closely related proteins (α, β, γ) encoded by distinct genes. α- and γ-adducin are expressed ubiquitously, while β expression is restricted to hematopoietic cells and the brain. In red blood cells (RBCs) adducin localizes to spectrin-actin junctions in the membrane skeleton as αβ heterotetramers. Previously (Gilligan et. al., PNAS, 1999) we showed that deletion of β-adducin results in osmotically fragile, microcytic RBCs and an overall phenotype of hereditary spherocytosis (HS). Notably, α-adducin was significantly reduced in β-adducin null RBCs. We also demonstrated that γ-adducin is present in low amounts in normal mouse RBCs and is upregulated ∼5-fold in β-adducin null RBCs. The increase in γ-adducin suggests that αγ heterotetramers may be compensating for the absence of β-adducin. In an effort to analyze γ-adducin function in RBCs in greater detail, we generated a conditional γ-adducin knockout allele in mice using a Cre-loxP strategy to delete exon 2 containing the start codon. All mice were maintained on a segregating B6.129 genetic background. Western blotting confirmed the absence of γ-adducin in spleen homogenates and RBC ghost preparations from γ-adducin null mice. All other membrane skeleton proteins examined by a combination of SDS-PAGE and western blotting, including α- and β-adducin, are normal in γ-adducin null RBCs (spectrin, ankyrin, band 3, protein 4.1, protein 4.2, dematin). Phenotypically, γ-adducin null mice display normal growth curves and show no overt defects. γ-adducin null RBCs appear normal on Wright’s stained peripheral blood smears and by scanning electron microscopy (SEM). The RBC count, hemoglobin content, hematocrit, MCV, reticulocyte %, osmotic fragility, and all other hematopoietic parameters are normal in γ-adducin null mice vs. wildtype. The apparent compensation by γ-adducin in β-adducin null red cells previously observed was tested by intercrossing mice null for γ- and β-adducin to produce βγ null double homozygotes. The additional loss of γ-adducin did not exacerbate the β-adducin null RBC phenotype as judged by examination of peripheral blood smears and SEM. Moreover, RBC osmotic fragility and complete blood counts in βγ-adducin null mice did not differ from β-adducin null mice. Western blotting of RBC ghost proteins confirmed reduction of α-adducin to ∼20% of normal in β-adducin null mice, as previously described. Strikingly, α-adducin in βγ-null RBC ghosts is reduced to barely detectable levels (

Description: Abstract 3186 Identification of new key players in erythropoiesis can lead to a better understanding of the etiology of anemia of unknown origin. Mouse models have significantly contributed to our understanding of normal erythropoiesis and the pathogenesis of erythroid disorders. Recently, we identified in the scat (severe combined anemia and thrombocytopenia) mouse model a missense mutation (G125V) in the Rasa3 gene, encoding a Ras GTPase activating protein (GAP). Homozygous scat mice present a cyclic phenotype with alternating episodes of crisis and remission. Crisis episodes are characterized by severe anemia and thrombocytopenia while, remarkably, the phenotype reverts to normal in the remission phase. Remissions are transient, however, and 94% of scat/scat mice die by P30 during a second crisis episode. We recently demonstrated a mechanism contributing to crisis episodes. The G125V mutation in Rasa3 is a loss of function mutation causing the protein to be mislocalized to the cytosol in scat reticulocytes. This results in loss of GAP activity and, as a consequence, increased levels of active GTP-bound Ras and a severe block in erythroid differentiation. Morpholino knockdowns of rasa3 in zebrafish result in profound anemia, confirming a conserved and non-redundant role for Rasa3 in vertebrate erythropoiesis. Here, we report that the cell cycle is affected in scat erythroid progenitors and extend studies to human primary erythroid cells. Using propidium iodine and flow cytometry, we found a significant increase in the G0/G1 phase (46.8% ± 3.1% in crisis vs 34.8% ± 2.5 in controls, × ± SD p

Description: Defects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes ofcno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3–independent mechanism critical to the biogenesis of lysosome-related organelles.

Description: Abstract 680 Scat (severe combined anemia and thrombocytopenia) is a spontaneous, autosomal recessive mutation coisogenic with the BALB/cBy inbred mouse strain. Homozygous scat mice present a cyclic phenotype with alternating episodes of crisis and remission. As its name implies, crisis episodes are characterized by severe anemia and thrombocytopenia, but significant lymphocyte depletion occurs as well. The first crisis episode begins in utero, lasts until postnatal day (P) 9 on average, and is associated with 10–15% mortality. Remarkably, in homozygotes that survive the first crisis, a remission phase occurs wherein the disease phenotype reverts to normal. This remission is transient, however, and is followed by a second crisis episode during which 94% of scat/scat mice die by P30. Previously we showed that the scat phenotype is transferrable via the hematopoietic stem cells and is also recapitulated in scat/scat, Hox11−/− double homozygotes in which a spleen does not develop, indicating that the splenic micro-environment plays little or no role in disease appearance or progression. Positional cloning of scat revealed a missense mutation in Rasa3 encoding a GTPase activating protein (GAP) that negatively regulates Ras function by accelerating GTP hydrolysis and converting Ras to the inactive GDP bound form. We further showed that Rasa3 is a conserved gene critical to vertebrate erythropoiesis via morpholino knockdowns in zebrafish which resulted in profound anemia. Here we report data that shed further light on RASA3 function during hematopoiesis. Overall, the data indicate that defects in RASA3 profoundly and negatively impact erythropoiesis and megakaryocytopoieis through, at least in part, a Ras-mediated mechanism. FACS analyses of scat spleen and bone marrow erythroid populations reveal a severe block in erythropoiesis during crisis periods. In the spleen, despite an initial increase in size due to expansion of Ter-119+ cells, there is ultimately a loss of compensatory erythropoiesis resulting in a return to normal cellularity and a striking loss of hemoglobinized cells as the crisis phenotype deepens. In addition, the bone marrow shows loss of Ter-119+ cells and overall cell depletion during crisis. Megakaryocyte numbers are increased in scat crisis BM and spleen. By transmission electron microscopy, scat crisis megakaryocytes display features characteristic of a significant developmental delay: a disorganized demarcation membrane system with no platelet forming areas and few granules with hypersegmented nuclei and excess rough endoplasmic reticulum. In addition to the severe anemia and thrombocytopenia, a significant lymphopenia occurs in scat crisis mice. However, the scat phenotype is not lymphocyte mediated, as the scat phenotype is completely recapitulated in mice doubly homozygous for scat and the immunodeficient mutations, scid and Rag1tm1Mom, in which B- and T-lymphocytes are completely depleted. Together these results suggest that lymphopenia is a secondary phenomenon in scat, and the severe anemia and thrombocytopenia aspect of the phenotype neither follows from nor is dependent upon loss of lymphocytes. Despite the delay observed in erythroid differentiation, some mature red cells are produced although ∼50% of these are reticulocytes. By confocal microscopy, we show that RASA3 protein localizes to the plasma membrane as well as internal membrane compartments in wild type reticulocytes, where it partially colocalizes with CD71. Western blot analyses of reticulocytes after Percoll gradient purification show that RASA3 is lost during the maturation step, both in vivo and in vitro. Interestingly, in scat, RASA3 is present in reticulocytes, but appears to be mislocalized, the protein being found in the cytosol. Preparation of ghosts from wild type and scat reticulocytes confirms that RASA3 is not attached to the membrane in scat animals during crisis. In pull-down assays active GTP-bound Ras is increased in scat crisis reticulocytes when compared to wild type, suggesting that scat is a RASA3 loss of function mutation due to its mislocalization and demonstrating a critical role for the RASA3-Ras axis during mammalian erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

Description: The membrane skeleton underlies the lipid bilayer and imparts strength and deformability to the red blood cell (RBC). Spectrin, the major component of the membrane skeleton, is crosslinked by short actin filaments into a two-dimensional array at junctional complexes, which are composed of multiple additional proteins including adducin. Three mammalian adducins (α, β, γ) exist that are encoded by distinct genes (Add1, Add2, Add3). α- and γ-adducin are ubiquitously expressed, with the highest expression of α-adducin seen in erythroid cells, brain, kidney, and heart. Expression of β-adducin is restricted to erythroid cells and brain. In RBCs heterotetramers of α- and β-adducin regulate actin filament length via barbed end capping. Previously, we deleted β-adducin in mice. Loss of β-adducin resulted in decreased α-adducin and up-regulated γ-adducin (5-fold), producing fragile, microcytic RBCs and an overall phenotype of hereditary spherocytosis (HS). Here, we report on recently generated α-adducin null mice. We inactivated the α-adducin gene (Add1) by targeted deletion of exons 10–12, and confirmed by western blotting the complete loss of α-adducin in RBC ghosts and in the brain. All mice were maintained on a segregating B6.129 genetic background. Heterozygous mice are normal in all parameters examined to date. Notably, although β-adducin gene expression is normal, western blotting revealed a complete absence of β-adducin protein in α-adducin null RBC ghosts. γ-adducin, present at low levels in normal mouse RBCs, was unchanged, as were all other membrane skeleton proteins examined by SDS-PAGE and/or western blotting (spectrin, ankyrin, band 3, protein 4.1, protein 4.2, dematin). α-adducin null mice display characteristics of mild compensated hemolytic anemia. The hematocrit is significantly decreased (43 vs. 46% in wildtype). RBCs are microcytic (MCV 41.9 vs. 46.4 fL) and osmotically fragile (50% lysis at 215 mOsm NaCl vs. 190 mOsm). The MCH is significantly decreased while the MCHC is significantly elevated, suggestive of RBC dehydration. The percentage of circulating reticulocytes is significantly increased (5.0 vs. 2.7%). Spleen weights are normal. Examination of peripheral blood smears and scanning electron microscopy confirms microcytic, anisocytotic RBCs with spherocytes and elliptocytes present. α-adducin null mice show postnatal growth retardation and approximately 66% develop critical hydrocephalus with marked expansion of the lateral and third ventricles by the fourth month of age. Normal littermates never show hydrocephalus. We conclude: α-adducin null mice have mild, compensated hemolytic anemia; β-adducin is unstable in RBCs in the absence of α-adducin; loss of α-adducin in the brain is associated with a high incidence of lethal hydrocephalus. Additional studies of the α-adducin null mouse model will be useful in defining protein functions and interactions in RBCs, the requirement for adducin in platelet function, the role of adducin in the brain, and its role in the regulation of systemic blood pressure.