Project description:The mitochondrial superoxide dismutase (SOD2) is a major antioxidant protein which detoxifies superoxide anion radicals generated by mitochondrial respiration (Weisiger and Fridovich, J. Biol. Chem. 1973). We designed a model of oxidative stress-induced anemia caused by SOD2-deficiency (Friedman et al. J. Exp. Med. 2001). Our previous work showed that mice reconstituted with SOD2-deficient hematopoietic stem cells develop an anemia with striking similarity to human sideroblastic anemia (SA) (Friedman et al. Blood 2004; Martin et al. Exp Hematol 2005). Our overall goal was to define early events in the pathogenesis of SOD2-deficiency SA and, in particular, to identify genes involved in the response of erythroid progenitors to oxidative stress. We compared gene expression of sorted TER-119+ CD71+ erythroblasts from SOD2-/- ('KO') versus Sod2+/+ ('WT') hematopoietic stem cell recipients using cDNA microarrays. Samples used in this study were re-hybridized with GLYCOv2 oligonucleotide arrays. GLYCOv2 oligonucleotide arrays are custom Affymetrix GeneChip arrays (Affymetrix, Santa Clara, CA) designed for the Functional Glycomics Gateway, collaboration between the Consortium for Functional Glycomics (CFG, http://www.functionalglycomics.org/) and Nature Publishing Group. A complete description of the array can be found at http://www.functionalglycomics.org/static/consortium/resources/resourcecoree.shtml . Hybridization results and quality control details can be found at https://www.functionalglycomics.org/glycomics/publicdata/microarray.jsp , by clicking on the âRaw Dataâ icon link for microarray experiment âJeff Friedman 1: Sod2 KO anemic miceâ. Briefly but importantly, one Sod2-/- sample, which was prepared separately, and whose GAPDH 3'/5' ratio was off range, was legitimately removed from the analysis of the GLYCOv2 and 430 2.0 arrays. Experiment Overall Design: We sorted Sod2-/- and Sod2+/+ size-matched mouse erythroblasts based on the expression of two developmental surface markers, CD71 and TER-119. Total RNA from 4 biological replicates per genotype were extracted and hybridized on 8 Affymetrix Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA). After quality controls (see scan protocol), analysis was performed with GeneSifter (VizX Labs, Seattle, WA) using 4 Sod2+/+ (control samples, WT) and 3 Sod2-/- (KO) replicates.

Project description:Gene expression profiling was performed using Anemic Sod 2 Knockout mice and was compared to Gene expression profiles from Sod 2 WT mice. Background: Our laboratory is interested in the role of oxidative stress in aging and human disease. Currently, a major focus of our efforts is the characterization of a murine hemolytic anemia resulting from oxidative stress, a model for the human disorder Sideroblastic Anemia. This model is based upon transplantation of hematopoietic stem cells from mice that are deficient in the antioxidant protein SOD2 (Friedman J. Exp. Med. 2001 193:925). SOD2 protects against the cytotoxicity of mitochondrial superoxide produced as a byproduct of oxidative phosphorylation (Weisiger J. Biol. Chem. 1973 248:4793). In characterization of the model to date, we have focused upon RBC proteomic analysis to identify 41 proteins differentially expressed when comparing membrane and soluble RBC fractions from SOD2-/- vs. SOD2+/+ mice (Friedman et al. submitted). In addition, we have characterized the in vivo response of animals to a novel catalytic antioxidant (Euk-189) that has b! oth SOD and catalase activities. In addition to proteins involved in mitochondrial function and stress response we find a number of cell surface molecules with significant differential expression. Thrombospondin is found at higher levels in SOD2-/- red cell membrane fractions. This is an extracellular matrix glycoprotein involved in cell adhesion and migration that interacts with glycosaminoglycans (especially the heparan sulfate proteoglycan perlecan) expressed at the endothelial cell surface (Feitsma J. Biol. Chem. 2000 275:9396, Vischer Eur. J. Cell. Biol. 1997 73:332, Sun J. Biol. Chem. 1989 15:2885). Thrombospondin is likely an acquired surface molecule on our red cells, which is of interest as this molecule has been found on the surface of sickle red cells and appears to mediate abnormal adhesion of red cells to vessel wall (Hillery Blood 1996 87:4879, Sugihara Blood 1992 15:2634). CD44 is also found at higher levels in SOD2-/- membrane fractions. CD44 is an adhesion receptor for the high molecular weight polysaccharide hyaluronic acid and has been shown recently to be involved in the trafficking of stem cells to the bone marrow (Avigdor Blood January 2004). Higher expression of these molecules on the surface of SOD2-/- red cells may affect their adhesive interactions with vascular endothelium and may in part explain the early removal of these cells from the circulationóresulting in the observed hemolytic anemia. Some proteins that accumulate on the surface of SOD2 deficient red cells are likely molecular damage sensors, binding to surfaces altered by oxidative damage. The role of specific glycoproteins in such binding remains to be elucidated. Additional surface proteins from red cells show differential expression, many of which are also glycosylated. Planned Experiment: As the next step in characterization of the anemia model, we will compare the gene expression profile of red cell progenitors isolated from Sod2-/- and Sod2+/+ animals. We have defined cell-sorting conditions for isolation of erythroid progenitor cells from marrow of our mice.

Project description:Echinoderm microtubule (MT)-associated protein-like 1 (Eml1) is mutated in the HeCo mouse, which exhibits subcortical band heterotopia (SBH), a developmental malformation of the cerebral cortex. EML1 mutations are also found in human patients affected by severe ribbon-like heterotopia, associated with epilepsy and intellectual disability (1). Neural progenitors in the ventricular zone (VZ) of the developing cerebral cortex undergo precisely regulated divisions, and mitotic perturbations contribute to pathological mechanisms (2). Eml1 is expressed in the mouse VZ and ectopic progenitors are present in the mutant developing cortex, when Eml1 is absent, at early stages of development (1). Thus, Eml1 is likely to play a role in neural progenitors during cortical development. We performed cell and molecular biology assays aiming to elucidate the function of Eml1 in neural progenitors, and explored the VZ of the HeCo mutant in order to find morphological perturbations that might explain the initiation of SBH formation (3). As part of this study, we searched for Eml1 molecular partners by pull-downs from mouse cortical extracts at embryonic day E13.5 and mass spectrometry (MS) analyses in order to identify the molecular pathways in which the protein is involved (3). Eml1 is formed by an N-terminal region that contains a dimerization domain and a C-terminal region that forms a ‘tandem atypical propeller in EMLs’ (TAPE) domain. The isolated N-terminal domain strongly binds MTs, while the C-terminal domain preferentially binds tubulin, and its beta-propeller structure is thought also to mediate the interaction with other molecules (4,5). We focused for this study on the N-terminal part of the protein (amino acids 1-178). Future study will validate or elucidate new interactions by using the C-terminal domain and/or the full-length protein. The results obtained from the N terminal MS data, integrated with other experimental findings, allow us to insert Eml1 in a network of proteins that are likely to regulate the assembly and function of the mitotic spindle in neural progenitors (3). More precisely, we showed that the protein regulates MT dynamics and its loss leads to perturbations in metaphase spindle length, which in turn impact progenitor morphology and behavior (3). References 1. Kielar, M., et al. Mutations in Eml1 lead to ectopic progenitors and neuronal heterotopia in mouse and human. Nat. Neurosci. 17, 923–933 (2014). 2. Bizzotto, S., & Francis, F. Morphological and functional aspects of progenitors perturbed in cortical malformations. Front Cell Neurosci. 9, 30; 10.3389/fncel00030 (2015). 3. Bizzotto, S., et al. Eml1 loss impairs apical progenitor spindle length and soma shape in the developing cerebral cortex. 4. Richards, M. W., et al. Crystal structure of EML1 reveals the basis for Hsp90 dependence of oncogenic EML4-ALK by disruption of an atypical β-propeller domain. Proc. Natl. Acad. Sci. U.S.A. 111, 5195–5200 (2014). 5. Richards, M. W., et al. Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain. Biochem. J. 467, 529–536 (2015).

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Comparative global gene expression of primary neural progenitors and MEFs transdfferentiated with OCT4 and SOD2

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