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

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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.

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Acknowledgments

We would like to thank Angela Welford for performing electron microscopy; Drs. Jane Rodman, Angela Wandinger-Ness and Vojo Deretic for sharing their expertise in the analysis of HIF-1α subcellular distribution. This study was supported by NIH P20 RR15636, NIH/NINDS R21 NS064185, and AHA 09GRNT2290178. Confocal images were generated in the UNM Cancer Center Fluorescence Microscopy Facility supported as detailed on the webpage http://hsc.unm.edu/crtc/microscopy/instru.html.

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Authors and Affiliations

  1. Department of Neurosurgery, University of New Mexico Health Sciences Center, 1101 Yale Blvd, Albuquerque, NM, 87131-0001, USA

    Tamara Roitbak

  2. Center for Molecular Discovery, University of New Mexico Health Sciences Center, Albuquerque, NM, USA

    Zurab Surviladze

  3. Department of Neurosciences, University of New Mexico Health Sciences Center, Albuquerque, NM, USA

    Lee Anna Cunningham

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  1. Tamara Roitbak
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  2. Zurab Surviladze
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Correspondence to Tamara Roitbak.

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Supplementary Figure 1

Characterization 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)

Supplementary Figure 2

Characterization 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)

Supplementary Figure 3

A: 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)

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Roitbak, T., Surviladze, Z. & Cunningham, L.A. Continuous Expression of HIF-1α in Neural Stem/Progenitor Cells. Cell Mol Neurobiol 31, 119–133 (2011). https://doi.org/10.1007/s10571-010-9561-5

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