Cellular and Molecular Neurobiology

, Volume 31, Issue 1, pp 119–133

Continuous Expression of HIF-1α in Neural Stem/Progenitor Cells

  • Tamara Roitbak
  • Zurab Surviladze
  • Lee Anna Cunningham
Original Research

DOI: 10.1007/s10571-010-9561-5

Cite this article as:
Roitbak, T., Surviladze, Z. & Cunningham, L.A. Cell Mol Neurobiol (2011) 31: 119. doi:10.1007/s10571-010-9561-5

Abstract

Hypoxia-inducible factor-1 alpha subunit (HIF-1α) is a transcriptional activator mediating adaptive cellular response to hypoxia. Normally degraded in most cell types and tissues, HIF-1α becomes stable and transcriptionally active under conditions of hypoxia. In contrast, we found that HIF-1α is continuously expressed in adult brain neurogenic zones, as well as in neural stem/progenitor cells (NSPCs) from the embryonic and post-natal mouse brain. Our in vitro studies suggest that HIF-1α does not undergo typical hydroxylation, ubiquitination, and degradation within NSPCs under normoxic conditions. Based on immunofluorescence and cell fractionation, HIF-1α is primarily sequestered in membranous cytoplasmic structures, identified by immuno-electron microscopy as HIF-1α-bearing vesicles (HBV), which may prevent HIF-1α from degradation within the cytoplasm. HIF-1α shRNAi-mediated knockdown reduced the resistance of NSPCs to hypoxia, and markedly altered the expression levels of Notch-1 and β-catenin, which influence NSPC differentiation. These findings indicate a unique regulation of HIF-1α protein stability in NSPCs, which may have importance in NSPCs properties and function.

Keywords

HIF-1α Stabilization Ubiquitination Neural stem cells 

Supplementary material

10571_2010_9561_MOESM1_ESM.tif (2.3 mb)
Supplementary Figure 1Characterization of the pNSPCs. pNSPCs were grown on poly-L-ornithine/laminin coated coverslips (~2 × 104 cells per 12 mm diameter coverslips) in serum-free medium, in the presence of growth factors EGF and bFGF (A). At 3 days in culture, the growth factors were removed and the cells were allowed to differentiate for 7-8 days (B). Differentiation was assessed by confocal microscopy to identify cells expressing cell-type specific antigens. Antibodies: mouse monoclonal anti-nestin (1:1000, BD Pharmingen), mouse monoclonal (1:1000) anti-GFAP (Accurate Chem. & Sci. Corp.), goat polyclonal anti-doublecortin/Dcx (1:300, Santa Cruz Biotech), and mouse monoclonal anti-βIII tubulin/Tuj1 (1:300, Promega). pNSPCs plated in clonal density (8-10 cell/ml) formed neurospheres during multiple passages (not shown). The neurospheres differentiated upon withdrawal of growth factors (C, D). Bar =10 μm. (TIFF 2397 kb)
10571_2010_9561_MOESM2_ESM.tif (3.3 mb)
Supplementary Figure 2Characterization of the anti-HIF-1α antibody. Neural progenitor cells (A and left panel on B) and the coronal sections of the adult mouse brain SVZ (B, right panel) were immunostained with HIF-1α antibodies purchased from Chemicon (A) and R&D Surveyortm IC Intracellular HIF-1α immunoassay kit (B). C—Left panel: mouse brain coronal sections were immunostained with anti-HIF-1α antibody from R&D kit, followed by Cy3-conjugated streptavidin secondary antibody. Right panel: Preabsorbtion control: HIF-1α antibody was pre-incubated with recombinant HIF-1α polypeptide (Prospec Protein Specialists) for 30 min at RT. Mouse brain coronal sections were immunostained with preabsorbed HIF-1α antibody, followed by Cy3-conjugated streptavidin secondary antibody. D—Electron micrographs demonstrating HIF-1α in NSPC cytoplasm (left panel) and in the vicinity of Golgi complex (middle panel). Right panel: EM control immunostaining with streptavidin-gold only. Bar = 50 nm. E-NSPC lysate was probed with anti-HIF-1α antibodies: biotinilated antibody from R&D immunoassay kit, goat anti-mouse antibody from R&D, mouse monoclonal antibodies from Chemicon, Sigma and Novus Biologicals, in concentrations recommended by vendors. F—Endothelial cell (ECs), astrocyte (Astr) and NSPC lysates were subjected to immunoblot using anti-HIF-1α antibody from the R&D immunoassay kit. Concentration of total protein in each loaded sample was adjusted using Bradford protein assay. (TIFF 3359 kb)
10571_2010_9561_MOESM3_ESM.tif (4.2 mb)
Supplementary Figure 3A: Immunofluorescence analysis of HIF-1α expression in adult mouse brain neurogenic zones SVZ and SGZ. Coronal sections of 8 week-old mouse brains were immunostained with antibodies against HIF-1α (red), nestin, doublecortin (Dcx,), GFAP and Sox-2 (green). Orthogonal image projections were generated on a Zeiss LSM510 confocal imaging system. B, Upper panels: immunochemical detection of brain tissue hypoxia. Two month-old C57BL/6 mice were injected via lateral tail vein with 60mg/kg pimonidazole hydrochloride (Hypoxyprobe™-1 Plus Kit). 20 min after Hypoxyprobe™-1 administration, mouse brains were harvested, post-fixed and subjected to immunofluorescence staining using FITC-conjugated mouse monoclonal antibody (Hypoxyprobe, Inc, clone 4.3.11.3). B, Lower panels: The combination of Hoechst nuclear staining and DIC confocal images of the SVZ and SGZ. Abbreviations: LV—lateral ventricle, St—striatum, cc—corpus callosum, GCL—dentate granule cell layer, h—hilus. Bar: 20 μm. The images were acquired on a Zeiss LSM510 and Zeiss LSM10-METAconfocal imaging systems. (TIFF 4325 kb)

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Tamara Roitbak
    • 1
  • Zurab Surviladze
    • 2
  • Lee Anna Cunningham
    • 3
  1. 1.Department of NeurosurgeryUniversity of New Mexico Health Sciences CenterAlbuquerqueUSA
  2. 2.Center for Molecular DiscoveryUniversity of New Mexico Health Sciences CenterAlbuquerqueUSA
  3. 3.Department of NeurosciencesUniversity of New Mexico Health Sciences CenterAlbuquerqueUSA

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