The Journal of Neuroscience, April 20, 2005 • 25(16):4099–4107 • 4099 Neurobiology of Disease Brain-Specific Knock-Out of Hypoxia-Inducible Factor-1␣ Reduces Rather Than Increases Hypoxic–Ischemic Damage Rob Helton,1* Jiankun Cui,2* John R. Scheel,1* Julie A. Ellison,1 Chris Ames,1 Claire Gibson,2 Barbara Blouw,3 Ling Ouyang,1 Ioannis Dragatsis,4 Scott Zeitlin,5 Randall S. Johnson,3 Stuart A. Lipton,2 and Carrolee Barlow1 1Laboratory of Genetics, The Salk Institute for Biological Studies, and 2Center for Neuroscience and Aging, The Burnham Institute, La Jolla, California 92037, 3Molecular Biology Section, Division of Biology, University of California, San Diego, La Jolla, California 92093, 4Department of Physiology, The University of Tennessee, Health Science Center, Memphis, Tennessee 38163, and 5Department of Neuroscience, University of Virginia School of Medicine, Charlottesville, Virginia 22908 ␣ ␣ Hypoxia-inducible factor-1 (HIF-1 ) plays an essential role in cellular and systemic O2 homeostasis by regulating the expression of genes important in glycolysis, erythropoiesis, angiogenesis, and catecholamine metabolism. It is also believed to be a key component of the cellular response to hypoxia and ischemia under pathophysiological conditions, such as stroke. To clarify the function of HIF-1␣ in the brain, we exposed adult mice with late-stage brain deletion of HIF-1␣ to hypoxic injuries. Contrary to expectations, the brains from the HIF-1␣-deficient mice were protected from hypoxia-induced cell death. These surprising findings suggest that decreas- ing the level of HIF-1␣ can be neuroprotective. Gene chip expression analysis revealed that, contrary to expectations, the majority of hypoxia-dependent gene-expression changes were unaltered, whereas a specific downregulation of apoptotic genes was observed in the HIF-1␣-deficient mice. Although the role of HIF-1␣ has been extensively characterized in vitro, in cancer models, and in chronic preconditioning paradigms, this is the first study to evaluate the role of HIF-1␣ in vivo in the brain in response to acute hypoxia/ischemia. Our data suggest, that in acute hypoxia, the neuroprotection found in the HIF-1␣-deficient mice is mechanistically consistent with a predominant role of HIF-1␣ as proapoptotic and loss of function leads to neuroprotection. Furthermore, our data suggest that functional redundancy develops after excluding HIF-1␣, leading to the preservation of gene expression regulating the majority of other previously characterized HIF-dependent genes. Key words: apoptosis; carotid; hippocampus; hypoxia; neuron; transcription Introduction tutively expressed subunit, HIF-1␤, and a subunit that is tightly ␣ The ability of cells and an organism to adapt to periods of hypoxia regulated by the cellular O2 concentration, HIF-1 (Wenger, is important for their survival in both physiological and patho- 2002). HIF-1␣ is the primary mammalian transcription factor physiological states (Bunn and Poyton, 1996). Hypoxia inducible specifically regulated by hypoxia; thus, HIF-1␣ and the genes factor-1 (HIF-1) is a basic helix-loop-helix (HLH) heterodimeric regulated by it have been the center of intense research because of transcription factor that is thought to play a major role in the their potential as therapeutic targets for ameliorating the effects response to hypoxia (Semenza, 2000). HIF-1 consists of a consti- of ischemia in a clinical setting. HIF-1␣ regulates genes that are important in tissue survival, such as vascular endothelial growth factor (VEGF), which has Received Nov. 5, 2004; revised Feb. 17, 2005; accepted Feb. 18, 2005. been shown to induce vasculogenesis and angiogenesis in many This work was supported by the Frederick B. Rentschler Developmental Chair, National Institute of Neurological ␣ Disorders and Stroke Grant NS039601-04 (C.B.), National Institutes of Health (NIH) Grants F31 NS10860-01 (J.R.S.), organ systems. Although HIF-1 is essential for adaptation to PO1 HD29587, R01 EY05477, R01 NS47973, R01 NS44326, R01 NS46994, and R01 NS43242, an Ellison Medical low oxygen levels, it has also been shown in vitro to mediate Foundation Senior Scholar Award (S.A.L.), and Microarray Core support by NIH Cancer Core Grant CA014195 (L.O.). hypoxia-induced growth arrest and apoptosis (Goda et al., 2003). We acknowledge the following: David Lockhart for scientific insights and support of this project; David and Dan Genetic studies to evaluate more precisely the role of HIF-1␣ in Lockhart for programming of the BullFrog software; Information Management Consultants (McLean, VA) and Tera- data (NCR, Dayton, OH) for donating the Teradata database and programming of the Teragenomics database; the organism had been hampered as a complete deletion leads to Ingenuity Systems for donation of the software and for figure preparation; Donna Ferriero and Howard Federoff for embryonic lethality (Ryan et al., 1998). Additional studies using scientificinsightsandsupportofthisproject;KathyChungandEmilyAnnasforassistancewithQ-PCRanalysis;Peter animals with tissue and organ deletions during development have Wagner for the use of the hypoxia chamber; and GOTree for analysis and programming. Additionally, we thank C. also been difficult to interpret because of developmental abnor- Doane and Jamie Simon for assistance with preparation of this manuscript. *R.H., J.C., and J.R.S. contributed equally to this work. malities (Ryan et al., 1998; Tomita et al., 2003). Correspondenceshouldbeaddressedtoeitherofthefollowing:CarroleeBarlow,LaboratoryofGenetics,TheSalk To understand the function of HIF-1␣ in the adult brain in Institute for Biological Studies, 10100 North Torrey Pines Road, La Jolla, CA 92037, E-mail: [email protected] or response to acute hypoxia, we generated mice with a late-stage [email protected]; or Stuart A. Lipton, Center for Neuroscience and Aging, The Burnham Institute, 10901 deletion of HIF-1␣ in the brain and exposed them to acute hy- North Torrey Pines Road, La Jolla, CA 92037, E-mail: [email protected]. DOI:10.1523/JNEUROSCI.4555-04.2005 poxic injuries (Dragatsis and Zeitlin, 2000). Surprisingly, mice Copyright © 2005 Society for Neuroscience 0270-6474/05/254099-09$15.00/0 deficient for HIF-1␣ showed significantly less neuronal cell loss in 4100 • J. Neurosci., April 20, 2005 • 25(16):4099–4107 Helton et al. • HIF-1 ␣ Neuroprotection response to hypoxia than control mice using two different para- number of TUNEL-positive cells per total number of cells counted in digms of neuronal hypoxia. Furthermore, gene-expression data matched areas of five sections at 40ϫ magnification. revealed that functional redundancy exists in the adult mouse RNA isolation. Mice were killed by cervical dislocation and dissected as brain, and the predominant role of HIF-1␣ in acute hypoxia is described previously (Sandberg et al., 2000). Tissues were dissected and Ϫ proapoptotic. snap frozen on dry ice. Samples were stored at 80°C until the RNA was isolated using Trizol according to the instructions of the manufacturer. Bilateral common carotid artery occlusion. Mice were anesthetized and Materials and Methods kept in the supine position. Body temperature was maintained by a blan- Generation of mice. All animal procedures were performed according to ket that was servo controlled at 37 Ϯ 1°C by a rectal temperature probe protocols approved by The Salk Institute for Biological Studies and The during the surgery and 1 h after reperfusion. Animals monitored for Burnham Institute Animal Care and Use Committees. Mice with com- longer time periods showed no variations. The common carotid arteries plete deletion of the HIF-1␣ allele (HIF-1␣ ϩ/Neo) were generated by were occluded bilaterally for 75 min. Arterial blood gases and blood replacement with a neomycin resistance gene (NeoR) as described previ- pressure were measured before and during ischemia through the right ously (Ryan et al., 1998). Mice that carried the conditional loxP alleles, femoral artery. We monitored relative cerebral blood flow by laser Dopp- HIF-1␣ alleles flanked by loxP sites (HIF-1␣ F/F), were generated by en- ler flowmetry, as we described previously (Wang et al., 1998), and found ⌬/⌬ F/F gineering a loxP site in the first intron and a loxP-flanked NeoR cassette no significant difference between HIF-1␣ and HIF-1␣ mice. in the second intron as described previously (Ryan et al., 2000). R1ag#5 Seventy-two hours after the procedure, mice were perfused with 4% mice expressing cAMP response element (CRE) under the control of the paraformaldehyde. The brains were dissected and submerged in 4% calcium/calmodulin-dependent kinase CaMKII␣-cre promoter (CAM- paraformaldehyde overnight at 4°C. Coronal sections were cut through CRE) were provided by Dr. Ioannis Dragatsis (University of Tennessee, the forebrain at 50 ␮m intervals with a vibratome. The sections were Memphis, TN) (Dragatsis and Zeitlin, 2000). To generate mice with dried at room temperature for 2 h and stained using acid fusin and cresyl brain predominant HIF-1␣ deletion (HIF-1␣ ⌬/⌬), CAMCRE mice were violet as described previously (Fujii et al., 1997). The grading scale for bred to mice carrying the HIF-1␣ F/F alleles. neuronal damage was as described previously (Fujii et al., 1997): 0, nor- Genotyping. Tail snips were collected from mice at 2 weeks of age for mal; 1, a few (30%) neurons damaged; 2, many neurons (30–70%) dam- genotyping. Tails were collected into lysis buffer, and DNA was isolated aged; and 3, majority of neurons (70%) damaged. All experiments were as described previously (Barlow et al., 1996). Genotyping was performed performed with the investigator blinded as to the genotype of the animal. using PCR. HIF-1␣flox and wild-type (WT) alleles were detected using Microarray analysis. cDNA arrays were used to identify the time of the following primers: HIF-24, GCA GTT AAG AGC ACT AGT TG; maximum overall gene expression in response to a hypoxic insult. Based HIF-25, GGA GCT ATC TCT CTA GAC C; and HIF-26, TTG GGG TGA on our Northern blotting results, HIF-1␣ induction is maximal 1 h after AAA CAT CTG C.