Overview
Our research focuses on the formation and function of blood cells. We are currently investigating three areas highly relevant to human disease. We study mechanisms that drive the assembly of the red blood cell (RBC) membrane skeleton, a complex structure critical to RBC stability. Hemolytic anemia due to membrane skeleton defects is one of the most common inherited diseases among northern Europeans (1 in 2,000-3,000). We are researching the development of intracellular organelles critical to platelet function, which, when defective, cause platelet storage pool disease, the second most common cause of inherited bleeding in humans. Additionally, we are determining the complex genetic interactions that influence steady-state peripheral blood counts. The steady-state white blood cell count is a significant risk factor for disease severity and early mortality in sickle cell anemia.
Scientific report
Genetics of Blood Cell Development and Disease
Introduction
Our research focuses on the formation and function of blood cells. We study mechanisms that drive developmental assembly of the red blood cell (RBC) membrane skeleton, a complex multi-protein structure critical to RBC stability; the development of intracellular organelles critical to platelet function; and complex genetic interactions that influence steady-state peripheral blood counts. These studies are highly relevant to human disease. Hemolytic anemia due to membrane skeleton defects is one of the most common inherited diseases in Northern Europeans (1:2,000 to 1:3,000). Defects in organelle biogenesis cause platelet storage pool disease (SPD), the second most common cause of inherited bleeding in humans. The steady-state white blood cell (WBC) count is a significant risk factor for disease severity and early mortality in sickle cell anemia, while the baseline hemoglobin (Hgb) concentration and hematocrit (Hct) level are independent risk factors for cardiovascular disease.
The RBC membrane skeleton.
An underlying spectrin-based membrane skeleton supports the plasma membrane of most cells. In RBCs, the major component of the membrane skeleton is spectrin, which is present as tetramers of α- and β-subunits. Spectrin tetramers are cross-linked into a two-dimensional array by short actin filaments at so-called junctional complexes, which also include the protein adducin. Spectrin is attached to the overlying plasma membrane by ankyrin, which binds the spectrin array to the cytoplasmic domain of the integral membrane protein known as band 3. During RBC development, membrane skeleton components are synthesized asynchronously. According to the "sequential assembly" hypothesis, spectrin and other membrane skeleton components shift from the cytoplasm to the membrane only upon induction of band 3 synthesis and its insertion into the RBC lipid bilayer. Studies from our laboratory, however, dispute this hypothesis, as a membrane skeleton is assembled normally in band 3-null RBCs. Hence, some other mechanism must exist to direct skeleton assembly.
Adducin shows characteristics in vitro that make it an attractive candidate to direct membrane skeleton assembly in vivo. Three genes encoding α- (Add1), β- (Add2), and γ- (Add3) adducin exist, and all are expressed in mouse RBCs. A full assessment of the role of adducin in RBC development will require multiple targetings with interbreeding to produce double and triple homozygotes. We have successfully generated targeted null mutations for α-, β-, and γ-adducin in mice. As expected, each mutation by itself produces a mild blood phenotype due to compensation by the remaining subunits. In combination, however, the blood anemia phenotype is exacerbated. For example, combined βγ-null RBCs are more severely aberrant than are β-null RBCs. Our ultimate goal is to produce all combinations, including the triple homozygous null, in order to fully assess the developmental role of the adducins in erythropoiesis. Interestingly, in addition to its potential role in RBC membrane skeleton assembly, adducin has been implicated in the control of systemic blood pressure, although this role is controversial, and is also present in blood platelets. Our adducin knockout strains will allow us to assess the role of all three adducins in blood pressure control and in platelet function.
Platelet storage pool disease (Hermansky-Pudlak syndrome)
Platelet SPD causes excessive bleeding due to a lack of platelet-specific organelles termed dense bodies, which are required for formation of the platelet plug at the site of injury. In one of the most severe forms of SPD, Hermansky-Pudlak syndrome (HPS), defects in developmentally related organelles-melanosomes and lysosomes-result in albinism and lysosomal storage disease, respectively, in addition to the bleeding diathesis. In both humans and mice, HPS is genetically heterogeneous. We previously cloned the genes responsible for two mouse HPS mutations, cappuccino (cno) and reduced pigmentation (rp). By N-ethyl-N-nitrosourea (ENU) mutagenesis, several novel HPS mutations have been generated, as determined by their lack of dense bodies (the "gold standard" diagnostic for HPS) and coat pigmentation defects. Our future goals are to (1) complete the phenotyping, genetic mapping, and cloning of these novel HPS models; (2) determine the functions of the encoded gene products; and (3) identify genetic modifiers of HPS using a dominant suppressor/enhancer ENU mutagenesis strategy.
Genetic modifiers: QTL studies
In sickle cell disease, baseline WBC count is a strong predictor of disease severity, including development of acute chest syndrome, stroke, and early mortality. We are working to identify genetic modifiers of baseline WBC count in mice as predictors of modifying genes in humans. To date, six significant QTL for baseline WBC count have been identified. In addition, we have identified multiple QTL influencing baseline erythropoietic traits (e.g., RBC count, Hgb concentration, red cell volume, Hct), which are important risk factors for cardiovascular disease. We are currently working to narrow the critical chromosome intervals to facilitate future candidate gene analyses.
New mouse models
Phenotype-driven approaches such as the analysis of spontaneous or chemically-induced mutations are a powerful method to assign function to genes. Therefore, we continue to analyze spontaneous and ENU-induced mouse mutations showing anemia or platelet-dysfunction phenotypes. Multiple potential new HPS mutants and novel anemia mutants are in various stages of development.
Lab staff
Principal Investigator: Luanne L. Peters, Ph.D.
Research Scientists: Connie Birkenmeier, M.S., Raymond Robledo, Ph.D., Ken Sahr, Ph.D.
Research Assistant IV: Babette Gwynn, M.S.
Research Laboratory Manager: Steven L. Ciciotte, M.S.
Research Assistant III: Amy J. Lambert, B.S.
Visiting Investigators: Samuel E. Lux, IV, M.D., Adjunct Senior Staff Scientist, Kathryn M. John, M.S.
Research Administrative Assistant: Maxine Friend
Publication listings
Svenson KL, Ahituv N, Durgin RS, Savage H, Magnani PA, Foreman O, Paigen B, Peters LL. 2008. A new mouse mutant for the LDL receptor identified using ENU mutagenesis. J Lipid Res 49, 2452-2462.
Campanella ME, Chishti A, Wandersee NJ, Peters LL, Narla M, Gilligan D, Low PS. 2008. Characterization of glycolytic enzyme interactions with murine erythrocyte membranes in wild type and membrane protein knockout mice. Blood 112, 3900-3906.
Robledo RF, Ciciotte SL, Gwynn B, Sahr KE, Gilligan DM, Peters LL. 2008. Targeted deletion of a -adducin results in absent b - and g -adducin, compensated hemolytic anemia, and hydrocephalus in mice. Blood 112, 4298-4307.
Alper SL, Vandorpe DH, Peters LL, Brugnara C. 2008. Reduced DIDS-sensitive chloride conductance in Ae1-/- mouse erythrocytes. Blood Cells Mol Dis 41, 22-34.
Li R, Svenson KL, Peters LL, Churchill GA. 2008. The relationships of dietary fat, body composition and bone mineral density in inbred mouse strain panels. Physiol Gen 33, 26-32.
Svenson KL, Smith RV, Magnani PA, Suetin HR, Paigen B, Naggert JA, Churchill GA, Peters LL. 2007. Multiple trait measurements in 43 inbred mouse strains captures the phenotypic diversity characteristic of human populations. J App Physiol 102, 2369-78.
Peters LL. 2007. Spectrin unfolding mutations: kinks in the links. Blood 109:3133-3134.
Peters LL, Robledo RF, Bult CJ, Churchill GA, Paigen BJ, Svenson KL. 2007. The mouse as a model for understanding human biology: a resource guide for complex trait analysis. Nat Gen Rev 8, 58-69.
Stehberger P, Shmukler BE, Stuart-Tilley AK, Peters LL, Alper SL, Wagner CA. 2007. Distal renal tubular acidosis in mice lacking the AE1 (band 3) Cl-/HCO3 exchanger (Slc4a1). J Am Soc Nephrol 18:1408-1418.
Chen H, Khan AA, Liu F, Gilligan DM, Peters LL, Messick J, Haschek-Hock WM, Li X, Ostafin AE, Chishti AH. 2007. Combined deletion of mouse dematin-headpiece and beta-adducin exerts a novel effect on the spectrin-actin junctions leading to erythrocyte fragility and hemolytic anemia. J Biol Chem, 282:4124-35.
Pack AI, Galante RJ, Cater J, Maislin G, Metaxas D, Lu S, Zhang L, Smith RV, Kay T, Lian, J, Svenson KL, Peters LL. 2007. A novel method for high throughput phenotyping of sleep and wakefulness in mice with application to mutagenesis. Physiol Gen 28:232-238.
Soni S, Shashi B, Gwynn B, Sahr KE, Peters LL, Hanspal M. 2006. EMP null mice are non-viable and exhibit erythroid and macrophage differentiation defects. J Biol Chem 281:20181-20190.
Rivera A, De Franceschi L, Bize I, Peters LL, Gascard P, Mohandas N, Brugnara C. 2006. Effect of complete protein 4.1R deficiency on ion transport properties of murine erythrocytes. Am J Physiol 291, C880-C886.
Gwynn B, Smith RS, Rowe LB, Taylor BA, Peters LL. 2006. A mouse TRAPP related protein is involved in pigmentation. Genomics 88, 196-203.
Peters LL, Lambert AJ, Zhang W, Churchill GA, Brugnara C, Platt OS. 2006. Quantitative trait loci for baseline erythroid traits. Mamm Genome 17, 298-309.
Inoue Y, Peters LL, Yim SH, Inoue J, Gonzalez FJ. 2006. Hepatocyte nuclear factor 4a is critical for blood coagulation homeostasis. J Mol Med 84, 334-344.
Shaw GC, Cope JJ, Li L, Corson K, Hersey C, Ackermann GE, Wingert RE, Trede NS, Traver D, Barut BA, Gwynn B, Minet E, Donovan A, Brownlie A, Weiss ME, Peters LL, Zon LI, Kaplan J, Paw BH. 2006. Frascati, a mitochondrial transporter of iron for heme biosynthesis, is essential in developing erythroblasts. Nature 440, 96-100.
Peters LL, Zhang W, Lambert AJ, Brugnara C, Churchill GA, Platt OS. 2005. Quantitative trait loci for baseline white blood cell count, platelet count, and mean platelet volume. Mamm Genome 16, 749-763.
Yang SH, Shrivastav A, Kosinski C, Sharma RK, Chen MH, Berthiaume LG. Peters LL, Chuang P-T, Young, SG, Bergo MO. 2005. N-Myristoyltransferase 1 is essential for early mouse development. J Biol Chem 280:18990-18995.
Rabenstein RL, Addy NA, Caldarone BJ, Asaka Y, Gruenbaum LM, Peters LL, Gilligan DM, Fitzsimonds, RM, Picciotto MR. 2005. Impaired synaptic plasticity and learning in mice lacking beta-adducin, an actin-regulating protein. J Neurosci 25:2138-2145.
De Franceschi L, Rivera A, Fleming M, Honczarenko M, Peters LL, Gascard P, Mohandas N, Brugnara C. 2005. Evidence for a protective role of the Gardos channel against hemolysis in murine spherocytosis. Blood 106:1454-1459.
Gwynn B, Martina JA, Bonifacino JS, Sviderskaya EV, Lamoreux LM, Bennett DC, Moriyama K, Huizing M, Helip-Wooley A, Gahl WA, Webb LS, Lambert AJ, Peters LL.2004. Reduced pigmentation (rp), a mouse model of Hermansky-Pudlak syndrome, encodes a novel component of the BLOC-1 complex. Blood 104: 3181-3189.
Clark AT, Goldowitz D, Takahashi JS, Vitaterna MH, Siepkas SM, Peters LL, Frankel WN, Carlson GA, Rossant J, Nadeau J, Justice MJ. 2004. Implementing large-scale ENU mutagenesis screens in North America. Genetica 122: 51-64.
Peterson KR, Fedosyuk H, Zelenchuk L, Nakamoto B, Yannaki E, Stamatoyannopoulos G, Ciciotte S, Peters LL, Scott LM, Papayannopoulou T. 2004. Transgenic Cre expression mice for generation of erythroid-specific gene alterations. Genesis 39:1-9.
Peters LL, Swearingen RA, Andersen SG, Gwynn B, Lambert AJ, Li R, Lux SE, Churchill GA. 2004. Identification of quantitative trait loci that modify the severity of hereditary spherocytosis in wan, a new mouse model of band 3 deficiency. Blood 103: 3233-3240.
Johnson KR, Gagnon LH, Webb LS, Peters LL, Hawes NL, Chang B, Zheng QY. 2003. Mouse models of USH1C and DFNB18: phenotypic and molecular analyses of two new spontaneous mutations of the Ush1c gene. Human Mol Genet 12: 3075-3086.
Gridley DS, Nelson GA, Peters LL, Kostenuik PJ Bateman TA, Morony S, Stodieck LS, Lacey Dl, Simske SJ, Pecaut MJ. 2003. Effects of spaceflight in the C57BL/6 mouse II: Activation, cytokines, erythrocytes, and platelets. J App Physiol 94:2095-2103.
Pecaut MJ, Nelson GA, Peters LL, Kostenuik PJ. Bateman TA, Morony S, Stodieck LS, Lacey DL, Simske SJ, Gridley DS. 2003. Effects of spaceflight in the C57BL/6 mouse I: Immune population distributions. J App Physiol 94: 2085-2094.
Bruce LJ, Beckmann R, Ribeiro ML, Peters LL, Chasis JA, Delaunay J, Mohandas N, Anstee DJ, Tanner MJA. 2003. A band 3-based macrocomplex of integral and peripheral proteins in the red cell membrane. Blood 101:4180-4188.
Svenson KL, Bogue MA, Peters LL. 2003. Identifying new mouse models of cardiovascular disease: a review of high-throughput screens of mutagenized and inbred mice. J App Physiol 94:1650-1659.
Lee G, Spring FA, Parsons SF, Mankelow TJ, Peters LL, Koury MJ, Mohandas N, Anstee DJ, Chasis JA. 2003. Novel secreted isoform of adhesion molecule ICAM-4: Potential regulator of membrane-associated ICAM-4 interactions. Blood 101:1790-1797.
Ciciotte SL, Moriyama K, Gwynn B, Huizing MA, Gahl WA, Bonifacino JS, Peters LL. 2003. Cappuccino, a mouse model of Hermansky-Pudlak syndrome, encodes a novel protein that is part of the pallidin-muted complex (BLOC-1). Blood 101:4402-4407.
Paw BH, Davidson AJ, Zhou Y, Li R,, Pratt SJ, Trede NS, Brownlie A, Donovan A, Liao EC, Ziai JM, Drejer AH, Guo W, Kim CH, Gwynn B, Peters LL, Chernova MN, Alper SL, Zapata A, Wickramasinghe SN, Lee MJ, Lux SE, Fritz A, Postlethwait JH, Zon LI. 2003. Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency. Nat Genet 34:59-64.
