Neuroglobin is up-regulated by and protects neurons from hypoxic-ischemic injury

Yunjuan Sun, Kunlin Jin, Xiao Ou Mao, Yonghua Zhu, and David A. Greenberg*

Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, CA 94945

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 4, 2001 (received for review September 4, 2001) are oxygen-binding present in bacteria, embryos, seeded at 3 ϫ 105 cells per well on 24-well culture protists, fungi, plants, and animals. Their functions have diverged dishes precoated with poly-D-lysine, and grown in Eagle’s MEM widely in evolution, and include binding, transport, scavenging, (GIBCO͞BRL) with 5% horse serum and 5% FBS (4). Cultures detoxification, and sensing of gases like oxygen, nitric oxide, and were treated with 10 ␮M cytosine arabinoside on day 6 and used . Neuroglobin (Ngb) is a recently discovered on day 11, when Ͼ95% of cells expressed the neuronal marker, monomeric with high affinity for oxygen and preferential microtubule-associated 2. To induce , cultures localization to vertebrate brain. No function for Ngb is known, but were placed in a modular incubator chamber (Billups- its affinity for oxygen and its expression in cerebral neurons Rothenberg, Del Mar, CA) containing humidified 95% air͞5% suggest a role in neuronal responses to hypoxia or ischemia. Here CO (control), or humidified 95% N ͞5% CO (hypoxic), for we report that Ngb expression is increased by neuronal hypoxia in 2 2 2 0–24 h at 37°C, and then returned to normoxic conditions for the vitro and focal cerebral ischemia in vivo, and that neuronal survival after hypoxia is reduced by inhibiting Ngb expression with an remainder, if any, of 24 h (10). Both control and hypoxic cultures contained 30 mM glucose. Cell viability was assayed by incu- antisense oligodeoxynucleotide and enhanced by Ngb overexpres- ͞ sion. Both induction of Ngb and its protective effect show speci- bating cultures with 5 mg ml of 3-(4,5-dimethylthiazol-2-yl)-2,5- ficity for hypoxia over other stressors. We conclude that hypoxia- diphenyltetrazolium bromide (MTT) at 37°C for 2 h and mea- inducible Ngb expression helps promote neuronal survival from suring A at 570 nm in solubilized cells by using a Cytofluor Series hypoxic-ischemic insults. 4000 multiwell plate-reader (PerSeptive Biosystems, Framing- ham, MA). In some experiments, results were confirmed by trypan blue exclusion (TBE). he fate of neurons undergoing hypoxic or ischemic injury is regulated by transcriptional and posttranscriptional events T Western Blotting. Cell lysates were prepared as described (4) and that contribute to competing cell-death and cell-survival pro- ␮ ͞ grams (1, 2). Survival-promoting events include the transcrip- 100- g protein samples were electrophoresed on 12% SDS tional induction or posttranslational activation of neuroprotec- PAGE gels and transferred to poly(vinylidene difluoride) tive proteins like erythropoietin (3), vascular endothelial growth membranes. Membranes were incubated overnight at 4°C with factor (4), and heme oxygenase (5). In many cases, these are a rabbit polyclonal Ab against Ngb (1:2,000), which was hypoxia-inducible proteins that help to counteract the adverse produced by immunizing with a synthetic peptide correspond- effects of hypoxia or ischemia by increasing anaerobic metabo- ing to amino acids 35–50 (NH2-CLSSPEFLDHIRKVML- lism, tissue vascularity, or oxygen delivery (6). Another strategy COOH) of mouse Ngb, and affinity-purified by using a Sul- for promoting the survival of metabolically active tissues like foLink kit (Pierce). A horseradish peroxidase-conjugated anti- muscle or nerve may involve the tissue-specific expression of rabbit secondary Ab (Santa Cruz Biotechnology) and a intracellular oxygen-binding proteins that can enhance oxygen chemiluminescence substrate system (NEN) were used to extraction and intracellular diffusion, or neutralize reactive visualize the immunolabeled bands (4). oxygen species. Examples include , in the case of muscle (7), and invertebrate nerve myoglobins (8). Cytochemistry. Cultures were fixed with 4% paraformaldehyde Neuroglobin (Ngb) is a newly discovered vertebrate globin and incubated overnight at 4°C with one or more of the following that is expressed most abundantly in neurons (9). Ngb was primary Abs: rabbit polyclonal anti-Ngb (1:200), mouse mono- identified by searching murine and human expressed sequence clonal anti-neuronal nuclear antigen (NeuN) (1:200, Chemicon), tag databases for partial globin-like sequences, then cloned, and and rabbit polyclonal anti-17–20-kDa caspase-3 cleavage prod- sequenced to reveal a 151-aa protein with a predicted molecular Ϸ uct (1:100, New England Biolabs). The secondary Abs (all 1:200) mass of 17 kDa, which exists as a monomer. Human and were fluorescein isothiocyanate-conjugated goat anti-rabbit or murine Ngbs show 94% sequence identity at the amino acid level anti-mouse IgG (Vector Laboratories) and rhodamine- but limited homology to other known globins. For example, there conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch). is Ͻ21% sequence identity with vertebrate myoglobins and Controls for nonspecific binding included omitting primary (Fig. Ͻ25% identity with vertebrate . The protein that 1c) or secondary Abs. In addition, in some cultures, nuclei were most closely resembles Ngb (30% amino acid identity) is the Ј intracellular nerve myoglobin of the polychaete annelid worm labeled with 4 ,6-diamidino-2-phenylindole (DAPI) or DNA Aphrodite aculeata (8). strand breaks were labeled with the Klenow fragment of DNA Because Ngb is an oxygen-binding heme protein that is polymerase I (Roche) followed by rhodamine avidin D (Vector expressed preferentially in cerebral neurons, we investigated its possible involvement in neuronal responses to hypoxia or isch- emia. The results indicate that Ngb is induced by neuronal This paper was submitted directly (Track II) to the PNAS office. hypoxia and cerebral ischemia and protects neurons from hyp- Abbreviations: DAPI, 4Ј,6-diamidino-2-phenylindole; HIF-1, hypoxia-inducible factor-1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NeuN, neuronal nu- oxia in vitro, suggesting that Ngb may have a role in sensing or clear antigen; Ngb, neuroglobin; ODN, oligodeoxynucleotide; SNP, sodium nitroprusside; responding to neuronal hypoxia. TBE, trypan blue exclusion. *To whom reprint requests should be addressed. E-mail: [email protected]. Materials and Methods The publication costs of this article were defrayed in part by page charge payment. This Cortical Neuron Culture. Neuronal cultures were prepared from article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. the cerebral hemispheres of 16-day Charles River CD1 mouse §1734 solely to indicate this fact.

15306–15311 ͉ PNAS ͉ December 18, 2001 ͉ vol. 98 ͉ no. 26 www.pnas.org͞cgi͞doi͞10.1073͞pnas.251466698 Downloaded by guest on September 27, 2021 NEUROBIOLOGY

Fig. 1. Neuronal hypoxia and ischemia induce Ngb protein expression. (a) Representative Western blot showing increased Ngb expression in cultured cortical neurons maintained without oxygen for the indicated number of hours (Left). Panel beneath the Western blot shows Ngb mRNA expression over the same time course. Expression of the 17-kDa band (arrow) was quantified by computer densitometry (mean Ϯ SEM, n ϭ 3; *, P Ͻ 0.05 relative to0hbyt test) (Right). (b) Representative Western blots (n ϭ 3) showing increased Ngb expression in cultures treated for 24 h with 300 ␮MCo2ϩ or 100 ␮M Dfx (Left), but no change with 0.1 ␮M staurosporine (Stauro) or 500 ␮M SNP (Right). (c) Fluorescence labeling of cultured cortical neurons showing Ngb immunoreactivity (red) in the cytoplasm of cells that express the neuronal nuclear antigen NeuN (green) (Left). Segregation of Ngb expression (green) and DNA damage (detected by labeling with the Klenow fragment of DNA polymerase I, red) into distinct populations, corresponding to viable cells with large nuclei (DAPI staining, blue) and nonviable cells with shrunken nuclei (Center). Preabsorption of the Ab with authentic Ngb peptide antigen abolished immunolabeling (Right). (d) Representative sections from contralateral, nonischemic rat cerebral cortex (Left) and penumbra (Center) or core (Right) of ischemic cerebral cortex at 24 h. Immunostaining for Ngb shows increased Ngb expression in the penumbra; this increased staining is localized to the cytoplasm of normal-appearing, unshrunken cells with neuronal morphology (Center, Insets). Brown, anti-Ngb; blue, cresyl violet. [Original magnification, ϫ400 (c and Insets to d) and ϫ200 (d)].

Laboratories), as described (11). Fluorescence signals were (red), 470͞505 nm for fluorescein isothiocyanate (green), and detected with a Nikon E800 epifluorescence microscope by using 360͞400 nm for DAPI (blue). Results were recorded with a excitation͞emission wavelengths of 535͞565 nm for rhodamine Magnifire digital color camera (Optronics). To evaluate the in

Sun et al. PNAS ͉ December 18, 2001 ͉ vol. 98 ͉ no. 26 ͉ 15307 Downloaded by guest on September 27, 2021 Fig. 2. Decreased Ngb expression exacerbates hypoxic neuronal death. (a) Representative Western blot showing decreased Ngb expression compared to untransfected control cells (Con) in cultured cortical neurons treated with an antisense ODN directed against Ngb, but not with a sense ODN (Left). Ngb expression was quantified (mean Ϯ SEM, n ϭ 3) by computer densitometry (*, P Ͻ 0.05 relative to Con by t test) (Right). (b) Cell viability, measured by MTT absorbance or TBE, in cultures maintained for 12 h without oxygen or in the presence of 0.1 ␮M staurosporine (Stauro) or 200–400 ␮M SNP, under standard conditions (no ODN) or after treatment with 5 ␮M sense or antisense ODN, added 3 h before the onset of, and present throughout the toxic exposure (n ϭ 3–6). *, P Ͻ 0.05 relative to no treatment (t test). (c) Fluorescence labeling of cultured cortical neurons treated with Ngb antisense (Upper) or sense (Lower) ODNs, showing immunoreactivity for the 17–20-kDa caspase-3 cleavage product (Left, red), ODN fluorescence (Center, green), and the merged images (Right, yellow). (Original magnification, ϫ400). Antisense-transfected cultures show an increase in the proportion of neurons that exhibit caspase-3 cleavage compared to sense- transfected cultures, consistent with the antisense-mediated decrease in cell viability shown in b.

vivo expression of Ngb, immunohistochemistry was done on plastic dishes and maintained as described (13, 14). Full-length cerebral cortical sections from rats subjected to 90 min of focal mouse Ngb cDNA (9) was cloned into a pcDNA 3.1 plasmid with cerebral ischemia followed by 4–24 h of reperfusion (12), using cytomegalovirus promoter (CLONTECH). The recombinant the same anti-Ngb primary Ab described above (1:200) and a plasmid (pcDNA-Ngb) or vector alone (pcDNA) was transfected horseradish peroxidase-conjugated goat anti-rabbit secondary into HN33 cells for 48 h by using FuGENE 6 (Roche), followed Ab (1:1000, Santa Cruz Biotechnology). by screening with G418 (Life Technologies, Grand Island, NY). Overexpression of Ngb was confirmed by Western blot as Oligodeoxynucleotide (ODN) Treatment. A phosphorothioate anti- described above. sense ODN labeled with fluorescein at the 5Ј end and directed against the initial coding region of the target Ngb mRNA Results and Discussion Ј Ј (5 -TCCGGGCGCTCCAT-3 , from nucleotides 90–77) was de- Hypoxia Induces Ngb Expression. To test whether Ngb expression is signed based on the mouse Ngb sequence obtained from Gen- induced in hypoxic cerebral cortical neurons, we generated and Bank (accession no. NM022414). This and a sense sequence (5Ј-ATGGAGCGCCCGGA-3Ј, from nucleotides 77–90) were affinity-purified a rabbit polyclonal Ab against a synthetic pep- synthesized commercially (Operon Technologies, Alameda, CA) tide corresponding to amino acids 35–50 of mouse Ngb. This Ab and purified by HPLC. Cultures were transfected with ODNs labeled a band on Western blots prepared from mouse cortical (1–10 ␮M) by using FuGENE 6 (Roche), beginning 3 h before neuron cultures at the predicted relative molecular mass of the onset of hypoxia. Cultures were analyzed by Western blotting 17,000 (Fig. 1a). When cultures were deprived of oxygen for up and by MTT cell viability assay, and fluorescence microscopy to 24 h, expression of this protein increased, as did the abun- was used to confirm transfection and examine its relationship to dance of Ngb mRNA, consistent with transcriptional induction. ␮ ␮ cell death, using caspase-3 activation as a marker. Expression was also increased by 300 M CoCl2 and by 100 M deferoxamine (Dfx) (Fig. 1b), which enhance the expression of HN33 Cell Culture and Transfection. HN33 cells (passage number hypoxia-inducible genes, including the major hypoxia-signaling Յ20) were plated at 1 ϫ 105 cells per well on uncoated, six-well transcription factor, hypoxia-inducible factor-1␣ (HIF-1␣) (15).

15308 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.251466698 Sun et al. Downloaded by guest on September 27, 2021 Fig. 3. Overexpression of Ngb reduces hypoxic cell death. (a) Representative Western blot showing increased Ngb expression in cultured HN33 cells maintained without oxygen for the indicated number of hours (Left). Expression of the 17-kDa band (arrow) was quantified by computer densitometry (mean Ϯ SEM, n ϭ 3; *, P Ͻ 0.05 relative to0hbyt test) (Right). (b) pcDNA vector or Ngb-expressing recombinant plasmid (pcDNA-Ngb) was stably transfected into HN33 cells, and NEUROBIOLOGY the overexpression of Ngb protein in pcDNA-Ngb-transfected cultures was confirmed by Western blotting (Left). Ngb expression was quantified by computer densitometry (mean Ϯ SEM, n ϭ 5; *, P Ͻ 0.05 relative to Con by t test) (Right). (c) Cell viability, measured by MTT absorbance or TBE, in pcDNA- or pcDNA-Ngb-transfected cultures maintained for 8 h (Hyp8) or 24 h (Hyp24) without oxygen, or for 24 h in the presence of 0.1 ␮M staurosporine (Stauro) or 300 ␮M SNP. *, P Ͻ 0.05 relative to untransfected control cultures (t test). (d) Oxygen consumption in untransfected (control) and pcDNA- or pcDNA-Ngb-transfected HN33 cells (5 ϫ 106 cells in 1 ml of DMEM) measured by using a Clark oxygen electrode (Hansatech Instruments, Pentney King’s Lynn, U.K.). Each tracing is the average of three independent experiments. The slopes give oxygen consumption (nmol O͞ml͞min) and were not significantly different across conditions (control, 7.46 Ϯ 0.90; pcDNA, 9.24 Ϯ 1.39; pcDNA-Ngb, 11.87 Ϯ 2.31; P ϭ 0.18 by ANOVA, n ϭ 9).

In contrast to the effects of hypoxia, CoCl2, and Dfx, other were therefore neurons (Fig. 1c). In hypoxic cultures stained for stressors [including staurosporine and the NO donor, sodium Ngb and for DNA damage with the Klenow fragment of DNA nitroprusside (SNP)] did not increase Ngb expression (Fig. 1b), polymerase I (11), Ngb was expressed most prominently in suggesting the specific involvement of hypoxia-signaling path- undamaged (Klenow-negative) cells. Ngb immunostaining was ways in Ngb induction. abolished when the Ab was preabsorbed with authentic Ngb Immunocytochemistry with the same anti-Ngb Ab used for peptide antigen. Western blotting showed that Ngb was localized to the cytoplasm To determine whether Ngb expression was also increased by of cells that expressed the neuronal nuclear antigen NeuN and cerebral ischemia in vivo, we immunostained sections from

Sun et al. PNAS ͉ December 18, 2001 ͉ vol. 98 ͉ no. 26 ͉ 15309 Downloaded by guest on September 27, 2021 cerebral cortex of mice subjected to focal cerebral ischemia by Possible Mechanisms for Induction of Ngb. These results are con- occlusion of the middle cerebral artery for 90 min followed by sistent with a role for Ngb as a hypoxia-inducible neuroprotec- reperfusion for 4–24 h as described (12). These sections showed tive factor in hypoxic-ischemic injury. How hypoxia stimulates increased Ngb immunoreactivity in the cytoplasm of neurons Ngb expression is uncertain, although hypoxia can induce he- from the ischemic compared to the nonischemic hemisphere moglobin synthesis in invertebrates (18) and may act through (Fig. 1d). This increase was greatest in the ischemic penumbra HIF-1 to regulate ␤-globin gene expression during vertebrate and less pronounced in what would evolve into the ischemic core. development (19). The effects of CoCl2 and Dfx (Fig. 1b) are These findings demonstrate that Ngb is expressed in neurons and consistent with involvement of HIF-1 in hypoxic induction of that its expression is increased by hypoxia and ischemia, espe- Ngb expression, as is the observation that the 5Ј-untranslated cially in neuronal populations that are destined to survive. region of Ngb (GenBank accession number NM 022414) con- tains several copies of the consensus HIF-1-binding sequence Reducing Neuroglobin Expression Worsens Hypoxic Injury. To begin 5Ј-RCGTG-3Ј (20), located 2073, 1977, 1445, 1041, 985, 627, 522, to investigate the possibility that Ngb protects neurons from and 64 nucleotides upstream of the transcription initiation site. hypoxia, cultured neurons were transfected with a phosphoro- thioate antisense ODN directed against the initial coding region Possible Mechanisms for Protection by Ngb. The manner in which of the target Ngb mRNA (5Ј-TCCGGGCGCTCCAT-3Ј, from Ngb exerts its neuroprotective effect is also uncertain. One nucleotides 90–77) or with a control sense sequence (5Ј- possibility is that, like myoglobin in muscle, it may bind oxygen ATGGAGCGCCCGGA-3Ј, from nucleotides 77–90), both la- and facilitate its delivery to mitochondria (7). To evaluate this beled with fluorescein at the 5Ј end. Transfection efficiency, possibility, we used a Clark oxygen electrode (21) to compare measured in cultures transfected with the fluorescent Ngb oxygen consumption in control and Ngb-overexpressing HN33 Ϯ antisense ODN and counterstained with DAPI, was 96 1% cells (Fig. 3d). Oxygen consumption did not vary significantly ϭ (n 10). Western blots showed that Ngb protein expression was across conditions, indicating that Ngb does not increase the rate reduced in antisense-transfected compared to sense-transfected of oxygen consumption and arguing for a different mode of or untransfected control cultures (Fig. 2a). The antisense- neuroprotective action. In some respects, this is not surprising mediated reduction in Ngb expression was associated with a because oxygen supply does not normally limit oxygen consump- decrease in the viability of cultured neurons exposed to hypoxia, tion and because the affinity of Ngb for oxygen may be too high whether measured by MTT absorbance, which reflects mito- for it to release oxygen under physiological conditions (22), chondrial function and is an early and sensitive indicator of cell although this is disputed (23, 24). Alternatively, and also by injury in this model, or TBE, which relates to membrane integrity analogy to myoglobin, Ngb might scavenge NO (25), which has and declines with more advanced damage (Fig. 2b). In contrast been implicated in hypoxic-ischemic neuronal injury (26). How- to its effect in hypoxia, Ngb antisense had no effect on the ever, the failure of Ngb antisense to exacerbate and of Ngb toxicity of staurosporine or SNP. Fluorescence microscopy overexpression to protect against SNP toxicity in our model showed that many antisense-transfected cells, but few sense- argues against this mechanism. Additional possibilities are that transfected cells, co-expressed the 17–20-kDa caspase-3 cleavage Ngb might be involved in sensing hypoxia and triggering pro- product that is generated in neurons undergoing ischemic cell tective cellular responses thereto, or in detoxifying mediators of death (16) (Fig. 2c). hypoxic-ischemic injury other than NO, for both of which actions there is precedent among nonvertebrate globins (18). Increasing Neuroglobin Expression Lessens Hypoxic Injury. To test further the protective effect of Ngb in hypoxia, full-length mouse Conclusion Ngb cDNA (9) was cloned into a pcDNA 3.1 plasmid with The recent discovery of Ngb (9) and the results presented here cytomegalovirus promoter (pcDNA-Ngb) and transfected into provide evidence for the existence of a novel endogenous and stably expressed in HN33, an immortalized hippocampal neuroprotective mechanism. Understanding how Ngb and other neuronal cell line (17). These cells were chosen because they hypoxia-inducible proteins confer neuronal protection may help provided high transfection efficiency and because their response in the development of improved treatment for ischemic disorders to hypoxia is well-characterized (4, 13). Hypoxia increased the such as stroke. expression of Ngb in HN33 cells, with a time course similar to that observed in cultured cortical neurons (Fig. 3a). Transfection We thank T. Burmester and T. Hankeln for the Ngb plasmid used in this with pcDNA-Ngb led to overexpression of Ngb (Fig. 3b) and study and D. Nicholls, S. Chalmers, B. Cochran, and G. del Rio for increased the viability of hypoxic HN33 cells, determined with helpful discussions and advice. This work was supported by the National either MTT or TBE (Fig. 3c). However, pcDNA-Ngb afforded Institute of Neurological Disorders and Stroke, National Institutes of no protection against staurosporine or SNP. Health, and by the Buck Institute for Age Research.

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