Ischemia induces different levels of hypoxia inducible factor-1α protein expression in interneurons and pyramidal neurons
© Ramamoorthy and Shi; licensee BioMed Central Ltd. 2014
Received: 18 March 2014
Accepted: 29 April 2014
Published: 5 May 2014
Pyramidal (glutamatergic) neurons and interneurons are morphologically and functionally well defined in the central nervous system. Although it is known that glutamatergic neurons undergo immediate cell death whereas interneurons are insensitive or survive longer during cerebral ischemia, the protection mechanisms responsible for this interneuronal survival are not well understood. Hypoxia inducible factor-1 (HIF-1) plays an important role in protecting neurons from hypoxic/ischemic insults. Here, we studied the expression of HIF-1α, the regulatable subunit of HIF-1, in the different neuronal phenotypes under in vitro and in vivo ischemia.
In a primary cortical culture, HIF-1α expression was observed in neuronal somata after hypoxia (1% oxygen) in the presence of 5 or 25 mM glucose but not under normoxia (21% oxygen). Interestingly, only certain MAP2-positive neurons containing round somata (interneuron-like morphology) co-localized with HIF-1α staining. Other neurons such as pyramidal-like neurons showed no expression of HIF-1α under either normoxia or hypoxia. The HIF-1α positive neurons were GAD65/67 positive, confirming that they were interneuron-type cells. The HIF-1α expressing GAD65/67-positive neurons also possessed high levels of glutathione. We further demonstrated that ischemia induced significant HIF-1α expression in interneurons but not in pyramidal neurons in a rat model of middle cerebral artery occlusion.
These results suggest that HIF-1α protein expression induced by ischemia is neuron-type specific and that this specificity may be related to the intracellular level of glutathione (GSH).
Neurons can be classified into three major groups, pyramidal neurons responsible for glutamate release, interneurons with round cell bodies responsible for γ–aminobutyric acid (GABA) release, and spiny interneurons with small cell bodies that can release both glutamate and GABA. Interestingly, ischemia-mediated vulnerability differs among neuronal subpopulations. Interneurons are resistant to ischemia in striatum, cortex, and hippocampus, whereas pyramidal neurons undergo immediate cell death under certain ischemic conditions [1–8]. However, the molecular mechanism for neuronal type-specific resistance to ischemia is not well understood.
Hypoxia inducible factor-1 (HIF-1) is a transcriptional factor that plays a critical role in cellular adaptation to low oxygen levels. It is a heterodimer consisting of two subunits, α and β. HIF-1β is constitutively expressed; the oxygen level has no effect on its expression. The protein level of HIF-1α is highly regulated by oxygen tension . Thus, the activity of HIF-1 is primarily determined by the expression of the subunit HIF-1α and not that of HIF-1β. During hypoxia, HIF-1α is stabilized, translocates to the nucleus, binds to HIF-1β, and initiates transcription. HIF-1 plays an important role in neuroprotection against ischemia by upregulating various growth factors such as vascular endothelial growth factor and erythropoietin. It has been shown that HIF-1α knockdown increases brain injury in a mouse model of transient focal cerebral ischemia . Inhibition of proline hydroxylase (PHD), an enzyme that initiates the degradation of HIF-1α, protects against glutamate-induced damage in the rat hippocampus . Moreover, it has been reported that HIF-1α expression can vary in different cells. For example, its stability and degradation is regulated in a cell-type-specific manner in carcinoma cell lines . The expression of HIF-1α differs in hepatoma and primary endothelial cells due to different degradation mechanisms . Degradation occurs mainly in the cytosol in HEPG2 cells and in both cytosol and nucleus in mouse brain endothelial cells . Previous data from our laboratory demonstrate that HIF-1α stability requires a reducing environment during ischemia and that increases in glutathione (GSH) levels stabilize HIF-1α in cortical neurons , indicating that protein levels of HIF-1α may vary among cells with different redox statuses.
We hypothesized that HIF-1α was expressed differently in pyramidal neurons and interneurons during hypoxic conditions. To test this hypothesis, we studied and compared the cell-type-specific expression of HIF-1α in pyramidal neurons and interneurons in a primary cortical neuronal culture exposed to hypoxia and an animal model of cerebral ischemia. We demonstrated that, under ischemic conditions, HIF-1α expression was remarkably stable in interneurons when compared to pyramidal neurons. HIF-1α stability in interneurons was consistent with an increase in intracellular GSH levels, suggesting that interneurons contain a highly reducing environment that maintains HIF-1 stability and expression during ischemia.
Materials and methods
Isolation of neurons
Primary neuronal cultures of cerebral cortices were obtained from Sprague–Dawley (SD) rat brains (postnatal day 0 [P0] to P3). Cultures were prepared according to Brewer et al.  with slight modifications. Whole cerebral cortices were dissected and then incubated for 50 min in 0.12% trypsin at 37°C. After the incubation, cells were washed completely with Hank’s balanced salt solution (HBSS) four times and dissociated with a fire-polished glass pipette in dissociation medium (HBSS, 0.1% BSA and 8 mM MgCl2), pelleted by centrifugation at 4000 g for 4 min at room temperature (RT), dissociated in starter medium (DMEM containing 10% FCS) and plated on coverslips. Coverslips were pretreated by incubation with poly-D-lysine (0.01%) for 1 hour (hr), then rinsed with sterile distilled water four times and dried before the cells were plated. Cultures were kept at 37°C in 5% CO2 for 1 hr, flooded with starter medium and incubated overnight at 37°C in 5% CO2. After 24 hrs, the medium was replaced with culture medium (Neurobasal plus 2 mM glutamine and B27 supplement). Every 4-6 days, half of the medium was replaced with fresh culture medium. After 12-18 days of in vitro culture (DIV), neuronal cells were used for the experiments.
In vitro hypoxia or cobalt chloride (CoCl2) treatment
Neuronal medium was removed and replaced with fresh, serum-free experimental medium (DMEM) containing 0, 5, or 25 mM glucose. For normoxia, neurons were incubated under 21% oxygen at 37°C for 3 hrs. For hypoxia, cells were maintained with 1% O2/5% CO2 balanced with N2 at 37°C for 3 or 5 hrs. Incubation periods were selected based on our previous studies . Expression of HIF-1α was observed in the cultured neurons after 3- and 5-hr hypoxic treatments, and further studies were performed only with 3-hr hypoxic treatments. For CoCl2 treatment, cortical neurons were incubated with 0.3 mM CoCl2 at 37°C for 3 hrs under normoxic conditions .
Middle cerebral artery occlusion (MCAO)
In vitro immunocytochemistry
Immunocytochemistry was performed as described by Ramamoorthy et al. . Briefly, cortical neurons were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 20 min at RT. Fixed cells were washed with PBS and permeabilized using 0.3% Triton X-100 in PBS for 15 min at RT and incubated with blocking solution (PBS containing 0.05% Triton X-100 and 0.25% BSA) for 30 min at RT. Then, neurons were incubated with specific primary antibodies overnight at 4°C. Cells were washed with blocking solution for 4 × 15 min and incubated with an appropriate secondary antibody for 90 min at RT in the dark. Coverslips were washed with blocking solution and mounted with the temporary mounting medium Vectashield H 1000 (Vector Laboratories, Burlingame, CA). Fluorescent intensity was quantified with Image-Pro Plus 5.1 (Media Cybernetics). We randomly selected several cell free areas and calculated mean intensity as background intensity. We then determined fluorescent intensity in neuronal soma in normoxic and hypoxic conditions. The background intensity was subtracted from neuronal HIF-1α intensity. Changes in HIF-1α intensity caused by hypoxia were normalized to the normoxic level.
In vivo immunocytochemistry
Rat brain was dissected after 90 min MCAO and 24 hr reperfusion. In vivo fixative was performed with 4% paraformaldehyde cardio perfusion. Then, brains were soaked in 4% paraformaldehyde overnight at RT and 30% sucrose for 48-72 hr at 4°C. Then, the brain was frozen using ice cold isopentane or liquid nitrogen for 2-3 min. Coronal brain sections were prepared from frozen brain sample using a cryostat. Slices were stained as described previously  with slight modifications. Briefly, after brain slices were washed with PBS and fixed (4% paraformaldehyde in PBS at RT) for 90 min, fixative was removed, and slices were washed with PBS and permeabilized in 1% Triton X-100 in PBS overnight at RT. Slices were incubated in blocking solution (PBS containing 2% BSA) overnight at 4°C and incubated for 48 hrs at 4°C with a primary antibody. Slices were washed in blocking solution for 15 min × 4 times and incubated with a secondary antibody for 4 hrs at RT. After washing, the slices were mounted in the temporary mounting medium Vectashield H1000. Control experiments were performed by omitting the primary antibody and incubating with secondary antibody alone.
Primary antibodies used were goat anti-HIF-1α (1-100; sc-8711; Santa Cruz Biotechnology), rabbit anti-HIF-1α (1-1000; 04-1006; Millipore Bioscience), mouse anti-HIF-1α (1-200; NB-100-123; Novus Biological), mouse anti-MAP2 (1:200; MAB378; Millipore Bioscience) and rabbit anti-GAD65/67 (1-400; AB1511; Millipore Bioscience). Secondary antibodies were donkey anti-goat Alexa 488 (1-100; Molecular Probes), goat anti-rabbit Alexa 488 (1-100; Molecular Probes), goat anti-mouse-FITC (1-50; Santa Cruz), donkey anti-mouse TRITC (1-50; Jackson ImmunoResearch) and donkey anti-rabbit DyLight 549 (1-2,500; Rockland). For double-staining experiments, antibodies were applied sequentially, starting with the anti-HIF-1α antibody. For antibody specificity, goat anti-HIF-1α antibody was incubated with HIF-1α blocking peptide (1-100; Santa Cruz) at RT for 2 hrs before staining the neurons for HIF-1α. Control experiments showed no significant bleed-through of the fluorescent labels or cross-reactivity between antibodies. As marked in Figure 1, the peri-infarct tissues in the ipsilateral cortex and the corresponding area in the contralateral cortex were selected as areas of interest. Images were obtained with a Leica DMI4000 microscope with a 40X objective and a Leica DFC340 FX Digital camera, using Leica LAS AF software. For quantification, the intensity of HIF-1α in the neuronal soma was randomly counted using MAP2- or GAD65/67-positive cell bodies. Fluorescent intensity was determined as described above.
GSH was detected by using monochlorobimane (MCB) as reported by Chatterjee et al. . Briefly, after hypoxic treatments, 0.1 mM of MCB was added directly to the neuronal petri dish and incubated for 20 min at 37°C. After the incubation, neurons were fixed and stained for specific neuronal markers using the method described above. The staining process was performed in the dark. MCB-GSH was visualized at an excitation wavelength of 380 nm and an emission wavelength of 480 nm using a fluorescence microscope. For quantification, MCB-GSH intensity was calculated for the neuronal soma. As it is impossible to measure MCB-GSH intensity in the same neurons before and after hypoxic treatments, different plates of neurons were treated with normoxia and hypoxia. Fluorescent intensity was determined as described above.
Dead or unhealthy neurons were counted visually by assessing morphological damage with the cytoskeletal protein MAP2 . Healthy cells were identified by intact somata and processes, unhealthy cells were differentiated by swelling or bulging in the MAP2-positive processes and dead neurons were identified by disruption/breakdown in the MAP2 staining in both the cell body and processes.
Image-Pro Plus, OriginPro7, and Excel were used for data analysis. One-way ANOVA and Student’s t-test were used for overall significance. Data are presented as the mean ± SD from at least 3 separate experiments. Differences were considered significant at p < 0.05.
HIF-1α stability after hypoxia/ischemia is neuron-type-specific
To further confirm these findings, we examined HIF-1α expression in different neurons in the cortex of a rat MCAO model. Double staining of HIF-1α and MAP2 in coronal brain slices revealed that not all MAP2-ir neurons expressed HIF-1α in the cortical area. Similar to our observation in primary cultures, neurons with a round-soma-like morphology showed HIF-1α-ir in the ipsilateral region (open arrow), but no HIF-1α staining was observed in the contralateral region (Figure 2D). In addition, neurons with a pyramidal-like morphology showed no HIF-1α expression (Figure 2D solid arrow), confirming the in vitro data.
Hypoxia-induced neuronal viability in the presence of different glucose concentrations
25 mM glucose
90.18 ± 3.00
5 mM glucose
89.63 ± 2.72
0 mM glucose
10.43 ± 5.02*
25 mM glucose
30.84 ± 1.58*
5 mM glucose
22.27 ± 5.37*#
0 mM glucose
8.02 ± 5.96*#
GAD65/67-positive neurons express HIF-1α following hypoxia/ischemia
GSH stabilizes HIF-1α in interneurons after hypoxia/ischemia
The present study demonstrates for the first time that HIF-1α expression is cell-type-specific among cortical neurons in response to hypoxic/ischemic insults. Specifically, certain interneurons express a significantly higher level of HIF-1α protein than pyramidal neurons. Furthermore, the present results reveal that reduction in the GSH level might play a role in decrease in the HIF-1α level of the interneurons.
Previous reports have shown that the two major classes of neurotransmitter-containing neurons, pyramidal neurons and interneurons, are differentially affected during ischemia [3, 21]. Consistent with these reports, our results demonstrate that hypoxia induces swelling in the somata or disruption of processes in pyramidal neurons. In contrast, the interneurons show intact somata and processes during hypoxia. Pyramidal neurons and interneurons have different mechanisms for buffering intracellular calcium during hypoxia [22, 23]. Interneurons contain specific calcium-binding proteins (e.g., parvalbumin (PV) and calbindin (CaBP)) to regulate intracellular calcium for the survival of these cells [24, 8]. Because calcium is a critical damaging factor in ischemic neuronal death, these calcium-binding proteins might contribute to interneuronal survival. However, Freund et al. found that there was no consistent and systematic relationship between neuronal CaBP or PV content and ischemic vulnerability . They and Crain et al. demonstrated that the majority of supragranular pyramidal cells that contained CaBP were the pyramidal cells most frequently degenerating after ischemia [26, 25]. In contrast, the density and distribution of non-pyramidal cells, mainly interneurons, containing CaBP or PV appeared qualitatively unchanged after ischemia [25, 26]. In addition, Larsson et al. have reported that increased neurotrophin signaling may provide neuroprotection in ischemic interneurons . Our results clearly demonstrate that among the cortical neurons, GAD65/67-positive neurons, but not pyramidal neurons, express HIF-1α following hypoxia. These results indicate a novel pathway that may contribute to the resistance of interneurons to ischemia. Consistent with this concept, HIF-1 has been found to regulate transcription of the NTRK2 gene, which stimulates neurotrophin signaling .
It is worth noting that CoCl2 treatments also induce significant expression of HIF-1α in interneurons, whereas minimal expression is seen in neurons containing pyramidal-like somata and apical dendrites with intact cell bodies and processes. The dose of CoCl2 used in the present experiments does not cause cell death in either interneurons or pyramidal neurons. This indicates that the expression of HIF-1α in hypoxic interneurons is not necessarily a result of cell survival.
Many factors can regulate HIF-1α protein stability in an oxygen-independent manner. For example, heat shock protein 90 can stabilize HIF-1α, and receptor for activated kinase C (RACK1) can lead to HIF-1α ubiquitination and degradation . Calcium might regulate HIF-1α expression in ischemic neurons. However, the available results are controversial or contradictory to each other. Calcineurin, a calcium-dependent protein phosphatase, may promote HIF-1α expression by de-phosphorylating RACK1 . In contrast, it has been reported that lowering the intracellular calcium concentration activates HIF-1 through inhibition of hydroxylation of HIF-1α . Unlike these results, Salnikow et al. reported that elevation of intracellular calcium neither induced the expression of HIF-1α protein nor stimulated HIF-1-dependent transcription . However, Mottet et al. argued that elevated calcium levels after prolonged hypoxia increased extracellular signal-regulated kinase 1/2 (ERK 1/2) activation and increased HIF-1 transcriptional activity but did not induce HIF-1α accumulation . Our results clearly show that GSH can stabilize HIF-1α protein in ischemic neurons. This is consistent with previous reports that HIF-1α can be strongly regulated by the redox environment [34–36], a mechanism that is of particular importance in the setting of ischemic brain injury because of the intrinsic changes in redox status. Although a more oxidizing environment has been suggested to stabilize HIF-1α in non-neuronal cells [37, 38], there is strong evidence supporting that a more reducing environment stabilizes HIF-1α, such as in COS7 cells , HeLa cells , HepG2 cells , MCF-7 cells , salmonid cells , renal medullary interstitial cells , and primary cultured neurons . Reactive oxygen species can increase the activity of both 26S and 20S proteasomal degradation pathways under hypoxic conditions [44, 45]. Thus, it is highly possible that the presence of high levels of GSH decreases the activity of the 20S and 26S pathways and stabilizes HIF-1α protein in interneurons. Based on this discussion, GSH may not be the only factor contributing to HIF-1α expression in the two neuronal types. Other antioxidants that are able to reduce reactive oxygen species in the neurons may also contribute to the stabilization of HIF-1α protein.
The exact mechanism by which hypoxic interneurons maintain higher levels of GSH than hypoxic pyramidal neurons remains unclear. However, the following mechanisms may be involved. First, interneurons may generate lower amounts of free radicals than pyramidal neurons in hypoxia/ischemia. It is well established that ischemia causes excess free radical generation, mainly via mitochondrial dysfunction caused by excessive calcium. Interneurons can have lower levels of NMDA receptor activity by inhibiting Cys-299 of the NMDA receptor subunit NR2A . This inactivation of glutamate receptors reduces the intracellular calcium and thus reduces free radical generation. Second, interneurons may have an enhanced defense system against oxidative stress. Interneurons have an enhanced and more efficient thioredoxin-2 system in detoxifying hydrogen peroxide, compared to pyramidal neurons . Enhanced expression of antioxidants such as superoxide dismutase and Bcl-2 has been observed in ischemic interneurons . In addition, the presence of nitric oxide may also help interneurons reduce free radical generation and protease activity [48, 46]. Overall, both low levels of free radical generation and enhanced levels of antioxidants may spare GSH in hypoxic interneurons.
We report here that expression of HIF-1α is mainly detected in interneurons during hypoxia/ischemia. High levels of GSH in interneurons may play a role in maintaining the cellular environment for the expression of HIF-1α. These results provide essential information for understanding the pathophysiology of cerebral ischemia.
PR is an assistant research professor at University of Kansas Medical Center. HS is an associate professor in pharmacology, toxicology and neuroscience at School of Pharmacy, University of Kansas.
Hypoxia inducible factor-1
Hank’s balanced salt solution
Middle cerebral artery occlusion
This research was supported in part by a grant from the National Institutes of Health (R01NS058807) and a Kansas University Center for Research startup fund.
- Monnerie H, Le Roux PD: Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro . Exp Neurol 2007, 205(2):367–382. doi:S0014–4886(07)00082–9 10.1016/j.expneurol.2007.02.007View ArticlePubMedGoogle Scholar
- Chesselet MF, Gonzales C, Lin CS, Polsky K, Jin BK: Ischemic damage in the striatum of adult gerbils: relative sparing of somatostatinergic and cholinergic interneurons contrasts with loss of efferent neurons. Exp Neurol 1990, 110(2):209–218. doi:0014–4886(90)90032-N 10.1016/0014-4886(90)90032-NView ArticlePubMedGoogle Scholar
- Frahm C, Haupt C, Witte OW: GABA neurons survive focal ischemic injury. Neuroscience 2004, 127(2):341–346. doi:10.1016/j.neuroscience.2004.05.027 10.1016/j.neuroscience.2004.05.027View ArticlePubMedGoogle Scholar
- Katchanov J, Waeber C, Gertz K, Gietz A, Winter B, Bruck W, Dirnagl U, Veh RW, Endres M: Selective neuronal vulnerability following mild focal brain ischemia in the mouse. Brain Pathol 2003, 13(4):452–464.View ArticlePubMedGoogle Scholar
- Aarts M, Liu Y, Liu L, Besshoh S, Arundine M, Gurd JW, Wang YT, Salter MW, Tymianski M: Treatment of ischemic brain damage by perturbing NMDA receptor- PSD-95 protein interactions. Science 2002, 298(5594):846–850. doi:10.1126/science.1072873 10.1126/science.1072873View ArticlePubMedGoogle Scholar
- Fryd Johansen F, Balslev Jorgensen M, Diemer NH: Resistance of hippocampal CA-1 interneurons to 20 min of transient cerebral ischemia in the rat. Acta Neuropathol 1983, 61(2):135–140. 10.1007/BF00697393View ArticlePubMedGoogle Scholar
- Johansen FF, Lin CT, Schousboe A, Wu JY: Immunocytochemical investigation of L-glutamic acid decarboxylase in the rat hippocampal formation: the influence of transient cerebral ischemia. J Comp Neurol 1989, 281(1):40–53. doi:10.1002/cne.902810105 10.1002/cne.902810105View ArticlePubMedGoogle Scholar
- Ferrer I, Soriano MA, Vidal A, Planas AM: Survival of parvalbumin-immunoreactive neurons in the gerbil hippocampus following transient forebrain ischemia does not depend on HSP-70 protein induction. Brain Res 1995, 692(1–2):41–46. doi:0006–8993(95)00527-W 10.1016/0006-8993(95)00527-WView ArticlePubMedGoogle Scholar
- Giaccia A, Siim BG, Johnson RS: HIF-1 as a target for drug development. Nat Rev Drug Discov 2003, 2(10):803–811. 10.1038/nrd1199View ArticlePubMedGoogle Scholar
- Baranova O, Miranda LF, Pichiule P, Dragatsis I, Johnson RS, Chavez JC: Neuron-specific inactivation of the hypoxia inducible factor 1 alpha increases brain injury in a mouse model of transient focal cerebral ischemia. J Neurosci 2007, 27(23):6320–6332. doi:27/23/6320 10.1523/JNEUROSCI.0449-07.2007View ArticlePubMedGoogle Scholar
- Batti L, Taylor CT, O’Connor JJ: Hydroxylase inhibition reduces synaptic transmission and protects against a glutamate-induced ischemia in the CA1 region of the rat hippocampus. Neuroscience 2010, 167(4):1014–1024. doi:10.1016/j.neuroscience.2010.03.011 10.1016/j.neuroscience.2010.03.011View ArticlePubMedGoogle Scholar
- Vordermark D, Katzer A, Baier K, Kraft P, Flentje M: Cell type-specific association of hypoxia-inducible factor-1 alpha protein accumulation and radiobiologic tumor hypoxia. Int J Radiat Oncol Biol Phys 2004, 58(4):1242–1250. doi:10.1016/j.ijrobp.2003.11.030 10.1016/j.ijrobp.2003.11.030View ArticlePubMedGoogle Scholar
- Zheng X, Ruas JL, Cao R, Salomons FA, Cao Y, Poellinger L, Pereira T: Cell-type-specific regulation of degradation of hypoxia-inducible factor 1 alpha: role of subcellular compartmentalization. Mol Cell Biol 2006, 26(12):4628–4641. doi:26/12/4628 10.1128/MCB.02236-05View ArticlePubMedPubMed CentralGoogle Scholar
- Guo S, Bragina O, Xu Y, Cao Z, Chen H, Zhou B, Morgan M, Lin Y, Jiang BH, Liu KJ, Shi H: Glucose up-regulates HIF-1alpha expression in primary cortical neurons in response to hypoxia through maintaining cellular redox status. J Neurochem 2008, 105(5):1849–1860. doi:JNC5287 10.1111/j.1471-4159.2008.05287.xView ArticlePubMedGoogle Scholar
- Brewer GJ: Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J Neurosci Res 1995, 42(5):674–683. 10.1002/jnr.490420510View ArticlePubMedGoogle Scholar
- Tan XL, Huang XY, Gao WX, Zai Y, Huang QY, Luo YJ, Gao YQ: CoCl2-induced expression of p300 promotes neuronal-like PC12 cell damage. Neurosci Lett 2008, 441(3):272–276. doi:S0304–3940(08)00894-X 10.1016/j.neulet.2008.06.050View ArticlePubMedGoogle Scholar
- Rogers DC, Campbell CA, Stretton JL, Mackay KB: Correlation between motor impairment and infarct volume after permanent and transient middle cerebral artery occlusion in the rat. Stroke 1997, 28(10):2060–2065. 10.1161/01.STR.28.10.2060View ArticlePubMedGoogle Scholar
- Ramamoorthy P, Whim MD: Trafficking and fusion of neuropeptide Y-containing dense-core granules in astrocytes. J Neurosci 2008, 28(51):13815–13827. doi:28/51/13815 10.1523/JNEUROSCI.5361-07.2008View ArticlePubMedPubMed CentralGoogle Scholar
- Chatterjee S, Noack H, Possel H, Keilhoff G, Wolf G: Glutathione levels in primary glial cultures: monochlorobimane provides evidence of cell type-specific distribution. Glia 1999, 27(2):152–161. 10.1002/(SICI)1098-1136(199908)27:2<152::AID-GLIA5>3.0.CO;2-QView ArticlePubMedGoogle Scholar
- Cao F, Hata R, Zhu P, Takeda S, Yoshida T, Hakuba N, Sakanaka M, Gyo K: Delayed neuronal cell death in brainstem after transient brainstem ischemia in gerbils. BMC Neurosci 11: 115. doi:1471–2202–11–115
- Schlander M, Hoyer S, Frotscher M: Glutamate decarboxylase-immunoreactive neurons in the aging rat hippocampus are more resistant to ischemia than CA1 pyramidal cells. Neurosci Lett 1988, 91(3):241–246. 10.1016/0304-3940(88)90687-8View ArticlePubMedGoogle Scholar
- Pisani A, Calabresi P, Tozzi A, Bernardi G, Knopfel T: Early sodium elevations induced by combined oxygen and glucose deprivation in pyramidal cortical neurons. Eur J Neurosci 1998, 10(11):3572–3574. 10.1046/j.1460-9568.1998.00398.xView ArticlePubMedGoogle Scholar
- Pisani A, Bonsi P, Calabresi P: Calcium signaling and neuronal vulnerability to ischemia in the striatum. Cell Calcium 2004, 36(3–4):277–284. doi:10.1016/j.ceca.2004.02.010View ArticlePubMedGoogle Scholar
- Freimann FB, Crome O, Shevtsova Z, Bahr M, Kugler S: Evaluation of long-term upregulation of Calbindin D28K as a preventive approach for ischaemic stroke. Int J Stroke 5(4):319–320. doi:IJS446
- Freund TF, Buzsaki G, Leon A, Baimbridge KG, Somogyi P: Relationship of neuronal vulnerability and calcium binding protein immunoreactivity in ischemia. Exp Brain Res 1990, 83(1):55–66.View ArticlePubMedGoogle Scholar
- Crain BJ, Westerkam WD, Harrison AH, Nadler JV: Selective neuronal death after transient forebrain ischemia in the Mongolian gerbil: a silver impregnation study. Neuroscience 1988, 27(2):387–402. 10.1016/0306-4522(88)90276-XView ArticlePubMedGoogle Scholar
- Larsson E, Lindvall O, Kokaia Z: Stereological assessment of vulnerability of immunocytochemically identified striatal and hippocampal neurons after global cerebral ischemia in rats. Brain Res 2001, 913(2):117–132. doi:S0006–8993(01)02762–7 10.1016/S0006-8993(01)02762-7View ArticlePubMedGoogle Scholar
- Martens LK, Kirschner KM, Warnecke C, Scholz H: Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of the TrkB neurotrophin receptor gene. J Biol Chem 2007, 282(19):14379–14388. doi:10.1074/jbc.M609857200 10.1074/jbc.M609857200View ArticlePubMedGoogle Scholar
- Liu YV, Baek JH, Zhang H, Diez R, Cole RN, Semenza GL: RACK1 competes with HSP90 for binding to HIF-1alpha and is required for O(2)-independent and HSP90 inhibitor-induced degradation of HIF-1alpha. Mol Cell 2007, 25(2):207–217. doi:10.1016/j.molcel.2007.01.001 10.1016/j.molcel.2007.01.001View ArticlePubMedPubMed CentralGoogle Scholar
- Liu YV, Hubbi ME, Pan F, McDonald KR, Mansharamani M, Cole RN, Liu JO, Semenza GL: Calcineurin promotes hypoxia-inducible factor 1alpha expression by dephosphorylating RACK1 and blocking RACK1 dimerization. J Biol Chem 2007, 282(51):37064–37073. doi:M705015200 10.1074/jbc.M705015200View ArticlePubMedPubMed CentralGoogle Scholar
- Berchner-Pfannschmidt U, Petrat F, Doege K, Trinidad B, Freitag P, Metzen E, de Groot H, Fandrey J: Chelation of cellular calcium modulates hypoxia-inducible gene expression through activation of hypoxia-inducible factor-1alpha. J Biol Chem 2004, 279(43):44976–44986. doi:10.1074/jbc.M313995200 10.1074/jbc.M313995200View ArticlePubMedGoogle Scholar
- Salnikow K, Kluz T, Costa M, Piquemal D, Demidenko ZN, Xie K, Blagosklonny MV: The regulation of hypoxic genes by calcium involves c-Jun/AP-1, which cooperates with hypoxia-inducible factor 1 in response to hypoxia. Mol Cell Biol 2002, 22(6):1734–1741. 10.1128/MCB.22.6.1734-1741.2002View ArticlePubMedPubMed CentralGoogle Scholar
- Mottet D, Michel G, Renard P, Ninane N, Raes M, Michiels C: Role of ERK and calcium in the hypoxia-induced activation of HIF-1. J Cell Physiol 2003, 194(1):30–44. 10.1002/jcp.10176View ArticlePubMedGoogle Scholar
- Welsh SJ, Bellamy WT, Briehl MM, Powis G: The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis. Cancer Res 2002, 62(17):5089–5095.PubMedGoogle Scholar
- Salceda S, Caro J: Hypoxia-inducible factor 1alpha protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem 1997, 272(36):22642–22647. 10.1074/jbc.272.36.22642View ArticlePubMedGoogle Scholar
- Wang GL, Jiang BH, Semenza GL: Effect of altered redox states on expression and DNA-binding activity of hypoxia-inducible factor 1. Biochem Biophys Res Comm 1995, 212(2):550–556. 10.1006/bbrc.1995.2005View ArticlePubMedGoogle Scholar
- Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT: Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci U S A 1998, 95(20):11715–11720. 10.1073/pnas.95.20.11715View ArticlePubMedPubMed CentralGoogle Scholar
- Chandel NS, McClintock DS, Feliciano CE, Wood TM, Melendez JA, Rodriguez AM, Schumacker PT: Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing. J Biol Chem 2000, 275(33):25130–25138. 10.1074/jbc.M001914200View ArticlePubMedGoogle Scholar
- Carrero P, Okamoto K, Coumailleau P, O’Brien S, Tanaka H, Poellinger L: Redox-regulated recruitment of the transcriptional coactivators CREB-binding protein and SRC-1 to hypoxia-inducible factor 1alpha. Mol Cell Biol 2000, 20(1):402–415. 10.1128/MCB.20.1.402-415.2000View ArticlePubMedPubMed CentralGoogle Scholar
- Huang LE, Arany Z, Livingston DM, Bunn HF: Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit. J Biol Chem 1996, 271(50):32253–32259. 10.1074/jbc.271.50.32253View ArticlePubMedGoogle Scholar
- Liu Q, Berchner-Pfannschmidt U, Moller U, Brecht M, Wotzlaw C, Acker H, Jungermann K, Kietzmann T: A Fenton reaction at the endoplasmic reticulum is involved in the redox control of hypoxia-inducible gene expression. Proc Natl Acad Sci U S A 2004, 101(12):4302–4307. 10.1073/pnas.0400265101View ArticlePubMedPubMed CentralGoogle Scholar
- Nikinmaa M, Pursiheimo S, Soitamo AJ: Redox state regulates HIF-1alpha and its DNA binding and phosphorylation in salmonid cells. J Cell Sci 2004, 117(Pt 15):3201–3206.View ArticlePubMedGoogle Scholar
- Yang ZZ, Zhang AY, Yi FX, Li PL, Zou AP: Redox regulation of HIF-1alpha levels and HO-1 expression in renal medullary interstitial cells. Am J Physiol Renal Fluid Electr Physiol 2003, 284(6):F1207-F1215.View ArticleGoogle Scholar
- Shi H: Hypoxia inducible factor 1 as a therapeutic target in ischemic stroke. Curr Med Chem 2009, 16(34):4593–4600. doi:CMC - AbsEpub - 069 10.2174/092986709789760779View ArticlePubMedPubMed CentralGoogle Scholar
- Kong X, Alvarez-Castelao B, Lin Z, Castano JG, Caro J: Constitutive/hypoxic degradation of HIF-alpha proteins by the proteasome is independent of von Hippel Lindau Protein Ubiquitylation and the transactivation activity of the protein. J Biol Chem 2007, 282(21):15498–15505. 10.1074/jbc.M700704200View ArticlePubMedGoogle Scholar
- Bidmon HJ, Emde B, Kowalski T, Schmitt M, Mayer B, Kato K, Asayama K, Witte OW, Zilles K: Nitric oxide synthase-I containing cortical interneurons co-express antioxidative enzymes and anti-apoptotic Bcl-2 following focal ischemia: evidence for direct and indirect mechanisms towards their resistance to neuropathology. J Chem Neuroanat 2001, 22(3):167–184. doi:S0891–0618(01)00126–0 10.1016/S0891-0618(01)00126-0View ArticlePubMedGoogle Scholar
- Kudin AP, Augustynek B, Lehmann AK, Kovacs R, Kunz WS: The contribution of thioredoxin-2 reductase and glutathione peroxidase to H2O2 detoxification of rat brain mitochondria. Biochim Biophys Acta 2012, 1817(10):1901–1906. doi:10.1016/j.bbabio.2012.02.023 10.1016/j.bbabio.2012.02.023View ArticlePubMedGoogle Scholar
- Chiueh CC, Rauhala P: The redox pathway of S-nitrosoglutathione, glutathione and nitric oxide in cell to neuron communications. Free Radic Res 1999, 31(6):641–650. 10.1080/10715769900301211View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.