Upregulation of transcription fact or NRF2-me diated

ORIGINAL PAPER

Upregulation of transcription factor NRF2-mediated oxidative stress response pathway in rat brain under short-term chronic hypobaric hypoxia

Niroj Kumar Sethy &Manjulata Singh &

Rajesh Kumar &Govindasamy Ilavazhagan &Kalpana Bhargava

Received:10May 2010/Revised:6August 2010/Accepted:13September 2010/Published online:5October 2010#Springer-Verlag 2010

Abstract Exposure to high altitude (and thus hypobaric hypoxia)induces electrophysiological,metabolic,and morphological modifications in the brain leading to several neurological clinical syndromes.Despite the known fact that hypoxia episodes in brain are a common factor for many neuropathologies,limited information is available on the underlying cellular and molecular mechanisms.In this study,we investigated the temporal effect of short-term (0–12h)chronic hypobaric hypoxia on global gene expression of rat brain followed by detailed canonical pathway analysis and regulatory network identification.Our analysis revealed significant alteration of 33,17,53,81,and 296genes (p <0.05,<1.5-fold)after 0.5,1,3,6,and 12h of hypoxia,respectively.Biological processes like regulation,metabolic,and transport pathways are temporally activated along with anti-and proinflammatory signaling networks like PI3K/AKT,NF-κB,ERK/MAPK,IL-6and IL-8sig-naling.Irrespective of exposure durations,nuclear factor (erythroid-derived 2)-like 2(NRF2)-mediated oxidative stress response pathway and genes were detected at all time points suggesting activation of NRF2-ARE antioxidant defense system.The results were further validated by

assessing the expression levels of selected genes in temporal as well as brain regions with quantitative RT-PCR and western blot.In conclusion,our whole brain approach with temporal monitoring of gene expression patterns during hypobaric hypoxia has resulted in (1)deciphering sequence of pathways and signaling networks activated during onset of hypoxia,and (2)elucidation of NRF2-orchestrated antioxidant response as a major intrinsic defense mechanism.The results of this study will aid in better understanding and management of hypoxia-induced brain pathologies.

Keywords Brain .Hypobaric hypoxia .Expression profile .Gene expression .NRF2-ARE

Introduction

Mammals have evolved sophisticated physiological net-works to sense and respond to altered oxygen conditions and to maintain oxygen homeostasis at the tissue level.The partial pressure of oxygen in the inspired air falls down with the increase in altitude resulting in compromised supply of oxygen to the tissues.This situation of hypobaric hypoxia is a common sickness of high-altitude sojourners like skiers,trekkers,soldiers,astronomers,miners,guides,tourists,and porters.The brain is exquisitely sensitive to hypoxia as it consumes 20%of body ’s oxygen and is the first organ to be compromised in response to low oxygen levels (Sharp and Bernaudin 2004;Wilson et al.2009).Within second to minutes of reduced oxygen supply to brain,a drastic reorganization of channel activities (Ballanyi 2004;Lopez-Barneo et al.2004),rearrangement of metabolic pathways (Pettersen et al.1986;Hochachka et

Electronic supplementary material The online version of this article (doi:10.1007/s10142-010-0195-y)contains supplementary material,which is available to authorized users.

N.K.Sethy (*):M.Singh :R.Kumar :G.Ilavazhagan :K.Bhargava (*)

Peptide and Proteomics Division,

Defence Institute of Physiology and Allied Sciences,Lucknow Road,

Timarpur,Delhi 110054,India e-mail:nksethy@https://www.360docs.net/doc/a76017976.html, K.Bhargava

e-mail:kalpanab@dipas.drdo.in

Funct Integr Genomics (2011)11:119–137DOI 10.1007/s10142-010-0195-y

al.1996;Harding et al.2003;Liu et al.2006),massive neurotransmitter release(Richter et al.1999),and overall suppression of energy-consuming processes(Pettersen et al. 1986;Hochachka et al.1996;Hochachka and Lutz2001) has been observed.While on a longer term,modulation of the expression of genes and proteins implicated in metabolism,apoptosis,inflammation and maintenance of structure under the transcriptional control of HIF-1α,AP1, CREB,and nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB)has been observed (Lopez-Barneo et al.2001;Sharp and Bernaudin2004;Seta and Millhorn2004).

Assessment of alterations in the gene expression profile has become an established strategy for the genome-wide correlation of gene transcript levels with hypoxia-induced physiological responses in various cells and tissues(Seta and Millhorn2004;Zhou et al.2008).In recent years, several groups have used gene expression analysis to understand brain genomic response to hypoxia using in vitro and in vivo experimental models(Bernaudin et al. 2002a;Ganfornina et al.2005;Gustavsson et al.2007; Zhou et al.2008).Majority of the studies have been focused to identify molecular mechanisms of oxygen sensing and response in neuronal cell models,such as PC12,RN46A cells,and astrocytes while,limited gene expression studies have been conducted in animal models (Rupert2008).The animal studies have been conducted on hypoxia sensitive tissues like hippocampus(Zhou et al. 2008),cortex(Gustavsson et al.2007;Zhou et al.2008), and carotid body(Ganfornina et al.2005)revealing biased information.Moreover,information for temporal gene expression patterns of brain during short-term chronic hypoxia is lacking limiting the understanding about early events.Hence,there is an urgent need to understand the brain gene expression patterns,underlying signaling path-ways,and molecular networks for better understanding and management of altitude-induced brain pathologies.

In this study,we conducted a systematic temporal(0–12h)search for hypoxia responsive early genes in rat brain using Illumina Bead-array platform followed by detailed canonical pathway analysis and regulatory net-work identification.Significant alterations in the expres-sion of a number of genes involved in functions like neuroprotection,angiogenesis,metabolism,transport,and inflammation were observed.Further,the expression levels of12differentially expressed genes were confirmed by quantitative real-time polymerase chain reaction(RT-PCR)along with region-specific expression analysis of nine genes.Our study identified significant activation of transcription factor nuclear factor(erythroid-derived2)-like2(NRF2)-orchestrated antioxidant response in brain during short-term chronic hypobaric hypoxia which was further validated.Materials and methods

Animals and hypoxia exposure

Male Sprague–Dawley rats,weighing180–200g(10week old)were used in this study.The animals were housed in the Experimental Animal Facility of the institute under 12/12h dark/light cycle and were fed standard food,and distilled water ad libitum.Animals were exposed to hypobaric hypoxia,at an altitude of25,000ft(310mm Hg)in a specially fabricated animal decompression cham-ber.The normoxic control group was maintained at normobaric conditions(760mmHg).The temperature of the simulation chamber was maintained at32°C and humidity at60±5%.The rate of ascent was about 300m/min and so was the rate of descent to sea level.A continuous air flow of8L/min was maintained in the chamber during the period of exposure to prevent accumu-lation of exhaled air.All experimental protocols were approved by the Animal Use and Care Committee of Defence Institute of Physiology and Allied Sciences and were in accordance with the principles and guidelines of the American Physiological Society’s“Guiding Principles in the Care and Use of Animals”.

RNA isolation

Whole brain as well as brain regions(n=3)like cortex, hippocampus,amygdala,and cerebellum was collected from 0.5,1,3,6,and12h hypoxia exposed as well as control rats and were immediately frozen in liquid nitrogen and stored at ?80°C until required.Total RNA from the whole brain and brain regions was extracted using TRI reagent(Sigma, USA),treated with RQ1DNase(Promega,USA),and later purified with RNeasy Mini kit(Qiagen,USA).RNA quality was determined using2100Bioanalyzer(Agilent,USA)and was quantified using a spectrophotometer(NanoDrop, USA).

Microarray hybridization

Microarray expression profiles were obtained using Illumi-na’s high-performance BeadArray technology.As per manufacturer’s instruction,Illumina TotalPrep RNA ampli-fication/in vitro transcription kit(Ambion,USA)was used to amplify and generate biotinylated cRNA from500ng total RNA for hybridization with Illumina BeadChip array. After purification,biotinylated cRNA was quantified with NanoDrop1000spectrophotometer(Thermo Scientific, USA).Of the biotinylated cRNA from each brain sample, 750ng was individually hybridized on the RatRef-12 Expression BeadChip.After incubation at58°C for20h, the BeadChip was washed,blocked,and incubated at room

temperature with streptavidin-Cy3-containing solution according to the manufacturer’s instruction.Fluorescent signals were scanned with BeadStation500GX Genetic Analysis System(Illumina Inc.,USA).Image registration and data extraction were automatic with BeadStudio3.1.1 (Illumina Inc.,USA).

Data analysis

Microarray data were analyzed by BeadStudio Gene Expression Module 3.2.6and Genome Viewer 3.1.7 (Illumina Inc.,USA).False discovery checks were per-formed with the“compute false discovery rate”function of the BeadStudio.The Illumina’s Cubic-Spline normalization algorithm was applied to adjust sample signals to minimize the effects of variation arising from non-biological factors. Comparison between groups of samples was done with the t test differential expression algorithm(detection p value< 0.05).Gene Ontology(GO)analysis for characterizing the biological properties of gene products was performed with Expression Analysis Systematic Explorer to statistically test for significant overrepresentation of the GO Consortium category Biological Process.

Network identification and canonical pathway analysis

The genes significantly regulated by hypoxia at all time points(<0.05FDR)were individually analyzed using Ingenuity Pathways Analysis(IPA)software(Ingenuity Systems,https://www.360docs.net/doc/a76017976.html,)to generate networks,ca-nonical pathways and functions.A dataset containing gene identifiers and their corresponding expression values was uploaded into the software.Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base.A0.05-FDR threshold cutoff was set to identify genes whose expression was signifi-cantly differentially regulated.These genes,called focus genes,were overlaid onto a global molecular network developed from information contained in the database. Networks of these focus genes were then algorithmically generated based on their connectivity and assigned a score.

Quantitative RT-PCR analysis

Aliquots of total RNA used for microarray hybridization along with total RNA from brain regions were used for quantitative RT-PCR to confirm the results obtained with microarray.Individual total RNA(1μg)samples from three rats for each time-point and six rats for each brain regions were reverse transcribed into cDNA using high-capacity cDNA Reverse Trancription Kit(Applied Biosystems, USA).The TaqMan probes(Applied Biosystems,USA) used for qRT-PCR were as follows:Lipocalin2(Rn00590612_m1),serum/glucocorticoid regulated kinase 1(Rn00570285_m1),adrenomedullin(Rn00562327_m1), vascular endothelial growth factor A(Rn00582935_m1), heme oxygenase(decycling)1(Rn00561387_m1),nitric oxide synthase3(Rn02132634_s1),solute carrier family16 (monocarboxylic acid transporters),member3 (Rn00578115_m1),EGF-like-domain,multiple7 (Rn00594758_m1),arachidonate15-lipoxygenase (Rn00696151_m1),eukaryotic elongation factor-2kinase (Rn00564087_m1),epoxide hydrolase2,cytoplasmic (Rn00576023_m1),prominin1(Rn00572720_m1),and n u c l e a r f a c t o r,e r y t h r o i d d e r i v e d2,l i k e2 (Rn00477784_m1)along with the endogenous control rat GAPDH and tubulin.The qRT-PCR was performed in a96-well format,under standard cycling conditions and reagents for TaqMan Gene Expression Assays(Applied Biosystems, USA)using StepOnePlus real time PCR system(Applied Biosystems,USA).All samples were run in triplicate with both primer set and endogenous control.Differences in gene expression,expressed as fold change,were calculated using the2àCΔΔt method.

Western blotting

After each hypoxic exposure,the animals(n=3)were sacrificed by cervical dislocation and total brain samples were collected.Ten percent tissue homogenate was pre-pared in ice-cold lysis buffer[10mM Tris–HCl,100mM NaCl,0.1mM Dithiothreitol,1mM EDTA,0.1%NaN3, 100μg/ml PMSF and protease inhibitor cocktail (Sigma-Aldrich,USA),pH7.6].The homogenate was centrifuged at10,000rpm for10min at4°C and the supernatant was collected.The concentrations of proteins samples were measured with Bradford reagent(Sigma-Aldrich,USA).Equal amount of protein samples(50μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(10%SDS-PAGE)using a miniVE vertical gel electrophoresis apparatus(Amersham Bioscien-ces,USA).Protein was transferred to a polyvinylidene difluoride membrane(Millipore,USA)using a semi-dry Trans-Blot system(Amersham Biosciences,USA)and probed with anti-NRF2(1:500,Santa Cruz Biotechnology, USA),anti-HO-1(1:500,Santa Cruz Biotechnology,USA) and anti-β-ACTIN(1:5,000,Sigma-Aldrich,USA)primary antibodies.After an overnight incubation,the blots were washed for30min three times with wash buffer(PBS, 0.5%Tween-20)at room temperature and further incubated for2h with appropriate horseradish peroxidase-conjugated secondary antibodies(1:5,000,Santacruz Biotechnology). The membranes were washed thrice as mentioned above and visualized on X-ray films using chemiluminiscent peroxidase substrate(Sigma-Aldrich,USA).Autoradio-gram signals were captured using a gel documentation

system(Model Omega,Ultra Lum Inc.,USA)and quantitative densitometric analysis was done with the default software.Statistical analysis was performed using GraphPad Prism3(GraphPad Software,Inc.,USA). Student’s t test was used to evaluate statistical significance and a p value<0.05was considered as significant.Results were expressed as mean±D of three replicates.

Results

Identification of temporal gene expression patterns

in hypobaric hypoxia and ontologies

The primary aim of this study was to identify the hypoxia-regulated early genes in rat brain under chronic hypobaric hypoxia(25,000ft,310mmHg).To examine the effect of chronic hypoxia,we examined the global gene expression profiles of RNA samples from0.5,1,3,6,and12h hypoxia along with normobaric control brain samples on Illumina platform.Stringent data analysis was performed in order to ensure the genes identified through microarray analysis were truly differentially expressed.Among the22,523genes present on BeadChips,microarray analysis detected a total of108,286,1,020,1,800,and3,740differentially regulated genes at0.5,1,3,6,and12h of hypoxia exposure respectively (Fig.1a,Supplementary material1).Among the differential-ly regulated genes,74were upregulated and34were downregulated at0.5h of exposure,162were upregulated and124were downregulated at1h,538were upregulated and482were downregulated at3h,1,027were upregulated and773were down regulated at6h,1,856were upregulated and1,884were down regulated at12h of exposure.Of the differentially expressed genes26,12,37,67,and234genes were upregulated and7,5,16,14,and62genes were downregulated by1.5-folds after0.5,1,3,6,and12h of hypobaric hypoxia respectively(Fig.1b,Table1).Analysis of expression profiles of all the time points revealed that22 genes(17upregulated and five downregulated)are common differentially regulated(Fig.2).The genes that were upregulated at all the time points were LCN2,VERGE, NR1D1,ADAMTS1,NFKBIA,SGK,DDIT4,NDRG1,and PLAT while genes like DNMT3A and SERPINB6A were downregulated(Fig.2).

Biological processes affected by hypoxia were investi-gated with GO analysis of significantly regulated genes at all the time points.Table2shows the20most significant temporal regulated GO biological processes in the brain ordered by p value across all the time points.The GO processes like biological regulation,regulation of biological process,regulation of cellular process,negative regulation of biological process,and developmental process were regulated by onset of the hypoxia exposure(0.5h).Similarly,the GO processes like primary metabolic process, metabolic process,macromolecule metabolic process,cel-lular component organization and biogenesis,and estab-lishment of cellular localization were regulated only after 3h of hypoxia exposure.The6h exposure regulated GO biological processes like vesicle-mediated transport and secretory pathway(Table2).Taken together,the GO biological processes were differentially regulated depend-ing on the duration of hypoxia.

Signaling pathways and associated functions altered

by hypobaric hypoxia

To gain an understanding of functional cellular pathways and processes that may be coordinately regulated during early phases of chronic hypobaric hypoxia,all the significant differentially regulated genes at each time point were uploaded to IPA and visualized.The functions of associated networks detected at0.5h of exposure are immunological disease,gene expression,cellular growth,and proliferation. Similarly,the major functions for1h hypoxia are cellular assembly and organization,cell morphology,nervous system development and function followed by cellular assembly and organization,cellular movement,nervous system develop-ment and function,lipid metabolism,molecular transport,and small molecule biochemistry at3h.The functions of associated networks at6h of exposure are DNA replication, recombination,and repair,neurological disease,behavior and at12h are cellular compromise,gene expression,inflamma-tory disease,metabolic disease,genetic disorder,and neuro-logical disease(Supplementary material3).

From the significant functions at each time point,the molecular networks were visualized with the Ingenuity tool. The networks assembled with IPA profiling of genes differentially expressed at0.5,1,3,6,and12h of hypoxia exposure,including interacting partners are depicted (Fig.3a;Supplementary material4).Genes demonstrated to be differentially upregulated or downregulated after microarray analyses are colored in red and green respec-tively.Genes that are uncolored were not found to be significantly altered with the hypoxia exposure.The class function of each of the genes,including enzymes,growth factor,and receptors are depicted by nodal shape.The fold change in gene expression compared with control animals is shown below each color node in respective figures. Genes like JUN,FOS,ACTB represent the central nodes in the pathways detected after0–3h of exposure while genes like NFE2L2,STAT3,AGT,CAPS3,and CCND1represent the central nodes of pathway detected after6–12h of exposure along with JUN and FOS(Fig.3a,Supplementary material2).These data suggested that prolonged hypoxia exposure activates more number of signaling molecules and interacting pathways in brain.

We next assessed the canonical pathways after chronic hypobaric hypoxia exposure.The top three canonical path-ways at 0.5h of hypoxia exposure were IL-10signaling,tight junction signaling,and arachidonic acid metabolism;at 1h were dendritic cell maturation,VDR/RXR maturation,nico-tine,and nicotinamide metabolism;at 3h were notch signaling,NRF2-mediated oxidative stress,IL-8signaling;at 6h were notch signaling,MIF regulation of innate immunity,semaphoring signaling in neurons while NRF2-mediated oxidative stress,antigen presentation pathway,and semaphoring signaling in neurons were the most significant pathway at 12h (Table 3,Supplementary material 1).Temporal analysis of canonical pathways revealed that PI3K/AKT and NF κB signaling pathways were detected during 0.5–6h of exposure,glycolysis/gluconeogenesis and GABA receptor signaling pathways were detected after 1h,VEGF signaling after 3h and ERK/MAPK signaling after 6h of hypoxia exposure (Supplementary material 2).Several pathways like NRF2-mediated oxidative stress response,IL-6and IL-17signaling,acute phase response signaling,angiopoitein signaling,thrombin signaling were detected at all the time points.

Expression analysis of hypoxia regulated genes

To validate results obtained with the microarray data and to further asses the temporal and tissue-specific expression of the differentially regulated genes,we assessed mRNA

expression of 12genes by real-time PCR.Figure 4shows average mRNA expression of LCN2,SGK1,ADM ,VEGFA ,HO-1,NOS3,SLC16A3,EGFL7,ALOX15,EEF2K ,EPHX2,and PROM1after 0.5,1,3,6,and 12h of hypoxia exposure.The expression data was in good agreement with the microarray data for all the genes further validating the hypoxia-induced expression levels.From the microarray data followed by real-time validation,it was apparent that maximum expression was observed at 12h of hypoxia exposure for most of the genes (Table 1;Fig.4).So we assessed the expression of nine genes (LCN2,SGK1,ADM ,VEGFA ,NRF2,HO-1,NOS3,SLC16A3,EGFL7)exposed to 12h hypoxia in various brain parts viz.amygdala,hippocampus,cortex,and cerebellum.Hypobaric hypoxia exposure increased expression of all the studied genes in the four regions and the highest expression levels were observed for hippocampus and cortex regions (Fig.5).Protein expression analysis of NRF2and downstream targets

To validate the NRF2-mediated antioxidant response path-way (Fig.3b ),the protein expressio levels of NRF2and its downstream target HO -1was evaluated by western blot analysis..Figure 6depicts the western blot results of the two selected genes after 0.5,1,3,6,and 12h of hypoxia exposure along with β-ACTIN as loading control.The maximum expression of NRF2was observed after 3h of

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200400600800100012001400160018002000Exposure time (h)

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3h

6h

12h

0.5h

1h

3h

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Fig.1Hypobaric hypoxia ex-posure (25,000ft,310mm Hg)temporally regulates gene ex-pression in rat brain.a The total number of upregulated and downregulated genes detected after 0.5,1,3,6,and 12h of hypobaric hypoxia exposure (n =3,p <0.05).b The number of significant differential regulated genes (±1.5-fold,p <0.05)detected after 0.5,1,3,6,and 12h of hypobaric hypoxia ex-posure.The highest number of hypoxia regulated genes and significant differential genes were detected after 12h of exposure

Table1Genes differentially expressed by chronic hypoxia arranged in functional classes

Functional class/Gene name GenBank no Symbol Fold change(h)

0.501030612

Neurofunction and neuroprotective genes

Adrenomedullin NM_012715.1Adm 1.36 1.24 2.11 2.80 2.19 Plasminogen activator,tissue NM_013151.2tPA0.810.820.69 1.61 1.20 Stanniocalcin1NM_031123.2Stc1–0.75 1.36 2.14 1.69 TIMP metallopeptidase inhibitor1NM_053819.1Timp1–0.380.340.82 1.75 Lectin,galactose binding,soluble3NM_031832.1Lgals3–0.380.54 1.01 1.58 Annexin A2NM_019905.1Anxa2–0.380.55 1.12 1.86 Epithelial membrane protein1NM_012843.2Emp1–0.92 1.40 2.20 2.20 Nestin NM_012987.1Nes–– 2.23 1.92–Aspartoacylase NM_024399.1Aspa–– 1.21 1.57–Serine(or cysteine)peptidase inhibitor,clade E,member1NM_012620.1Serpine1–– 1.18 1.78–RNA binding motif(RNP1,RRM)protein3NM_053696.1Rbm3––– 1.53 1.86 Carboxypeptidase E NM_013128.1Cpe–––0.58 1.86 Neuronal regeneration related protein NM_178096.2Nrep–––– 2.18 Chemokine(C-X-C motif)receptor4NM_022205.3Cxcr4–––– 2.03 Ribosomal protein S9NM_031108.2Rps9?0.92?0.65?5.67–?5.53 Activity-regulated cytoskeleton-associated protein NM_019361.1Arc–?1.66?2.08?3.06?3.74 MAS1oncogene NM_012757.2Mas1–?0.47?0.55?0.78?1.69 Angiogenesis,blood vessel development,and blood pressure regulation

EGF-like-domain,multiple7NM_139104.1Egfl7 1.070.830.92 1.58 1.62 Fibronectin1NM_019143.2Fn10.57–0.89 1.84 1.80 Angiopoietin2NM_134454.1Angpt2–– 1.09 1.99 4.07 Vascular endothelial growth factor A NM_031836.2Vegfa–– 1.08 1.75 3.10 Nitric oxide synthase3,endothelial cell NM_021838.2Nos3–– 1.57 2.42 2.48 Placental growth factor NM_053595.2Pgf–– 1.14 2.12 2.42 Thrombomodulin NM_031771.2Thbd–– 1.20 1.560.96 Heparanase NM_022605.1Hpse–– 1.30 2.30 1.20 Collagen,type IV,alpha NM_001135009.1Col4a1––0.89 1.59 1.66 Epoxide hydrolase2,cytoplasmic NM_022936.1Ephx2––?0.30?0.39?4.65 Metabolism

Solute carrier family16,member3(monocarboxylic acid

NM_030834.1Slc16a3– 1.04 1.93 3.02 3.05 transporter4)

Pyruvate dehydrogenase kinase,isozyme4NM_053551.1Pdk4– 2.28 2.68 3.32 3.34 Glycerol-3-phosphate dehydrogenase1(soluble)NM_022215.2Gpd1–0.68 1.21 2.10 1.55 Lipolysis stimulated lipoprotein receptor NM_032616.1Lsr– 1.28 1.55 1.36–Phospholipase A1member A NM_138882.1Pspla1–– 1.10 2.03 2.83 Hexokinase2NM_012735.1Hk2––– 1.49 1.76 Dipeptidylpeptidase6NM_022850.1Dpp6––– 1.22 1.64 Caveolin2NM_131914.2Cav2–––0.74 1.74 Prominin1NM_021751.2Prom1––?1.44?2.43?2.75 Thyroid stimulating hormone,beta NM_013116.1Tshb?1.98–––?1.08 Transport

FXYD domain-containing ion transport regulator5NM_021909.1Fxyd5––0.85 1.570.86 Chloride intracellular channel4NM_031818.1Clic4––0.65 1.02 1.85 Solute carrier family6(neurotransmitter transporter,

NM_013034.3Slc6a4––– 2.99 1.86 serotonin),member4

Chloride intracellular channel2NM_001009651.1Clic2 1.85––––Solute carrier family4(anion exchanger),member1NM_012651.2Slc4a1 1.87––––

0.501030612 Solute carrier family6(neurotransmitter transporter,glycine),

NM_203334.1Slc6a5–––– 2.10 member5

Solute carrier family3,member1NM_017216.1Slc3a1–––– 2.76 Solute carrier family14(urea transporter),member1NM_019346.2Slc14a1–––– 3.90 Solute carrier family39(iron-regulated transporter),member1NM_133315.2Slc40a1–?0.60?1.26?1.71?1.17 Solute carrier organic anion transporter family,member1c1NM_053441.1Slco1c1––?0.81?1.77?1.83 Transcription factors and effectors

Activating transcription factor3NM_012912.1Atf3 1.75 2.14 1.75––Nuclear receptor subfamily4,group A,member3NM_017352.1Nr4a3––?0.52?1.20?2.22 Eukaryotic elongation factor-2kinase NM_012947.2Eef2k?3.34?3.12?2.50–?0.95 Early growth response1NM_012551.2Egr1–––?1.51?2.07 FBJ osteosarcoma oncogene NM_022197.2Fos–––?1.56?1.01 Early growth response2NM_053633.1Egr2––––?2.28 Antioxidant Response

Metallothionein1a NM_138826.4Mt1a– 1.80 2.84 3.89 4.24 Heme oxygenase(decycling)1NM_012580.2HO-1–– 1.13 1.96 3.07 DNA-damage-inducible transcript4NM_080906.1Ddit40.88 1.16 1.62 1.64 1.10 Transferrin receptor protein1NM_022712.1Tfrc–––– 2.13 Apoptosis

TIMP metallopeptidase inhibitor3NM_012886.2Timp3––– 1.61 1.72 Caspase4,apoptosis-related cysteine peptidase NM_053736.2Casp11–––– 2.66 Arachidonate15-lipoxygenase NM_031010.2Alox15 4.41––?0.56?0.77 BCL2/adenovirus E1B19kDa-interacting protein3NM_053420.2Bnip3–?0.58?0.49–?2.57 B-cell leukemia/lymphoma2related protein A1d NM_133416.1Bcl2a1––?1.72?1.99–Immunogenic response

FK506binding protein5NM_001012174.1Fkbp5?0.76– 1.00 1.590.82 RT1class Ia,locus A1NM_001008827.1RT1-A1–––0.29 1.53 RT1class I,locus T24,gene4NM_001008826.3RT1-149–––0.72 1.60 CD93molecule NM_053383.1C1qr1––– 1.18 2.25 Integrin,beta2NM_001037780.2Itgb2–––– 2.90 Defensin NP-4precursor NM_173299.1Np4––––7.33 Defensin beta1NM_031810.1Defb1––?0.56?1.52?0.91 Inflammation

Tumor necrosis factor receptor superfamily,member12a NM_181086.2Tnfrsf12a– 1.55 1.330.92 1.11 Zinc finger protein36NM_133290.3Zfp36–0.76 1.41 1.670.74 Lectin,galactose binding,soluble3NM_031832.1Lgals3–0.380.54 1.01 Haptoglobin NM_012582.2Hp––– 2.39 2.09 Secreted phosphoprotein1NM_012881.2Spp1–––– 2.22 Prostaglandin-endoperoxide synthase2NM_017232.3Ptgs2––?0.68?0.68?1.51 Novel genes

Lipocalin2NM_130741.1Lcn27.37 6.87 5.490.3411.94 Vascular early response gene protein NM_001003403.1Verge 2.61 1.82 2.52 2.58 1.56 Cathepsin E NM_012938.1Ctse 2.51– 1.34 1.36 1.65 A disintegrin-like and metallopeptidse(reprolysin type)with

NM_024400.2Adamts1 1.270.73 1.55 2.05 2.72 thrombospondin type1motif,1

Retinal pigment epithelium65NM_053562.1Rpe650.750.82 1.18 1.76 1.66 Serine(or cysteine)proteinase inhibitor,clade B,member1a NM_001031642.1Serpinb1a 1.000.900.81– 1.68 Interferon induced transmembrane protein3NM_001136124.1Ifitm3–0.320.460.94 1.75

0.501030612

Kinesin family member22NM_001009645.1Kif22–0.320.460.94 1.75 Transgelin NM_001013127.1Tagln2––0.74 1.26 1.81 C-type lectin domain family4,member a3NM_001005891.1Dcir3–– 1.16 1.13 1.63 Carbonic anyhydrase12NM_001080756.1Car12––– 2.02 2.44 RAB31,member RAS oncogene family NM_145094.2Rab31––– 1.35 2.09 Phosphoglycerate mutase family member5NM_001025272.1Pgam5––– 1.27 1.85 Proopiomelanocortin NM_139326.2Pomc–––– 2.07 Abhydrolase domain containing5NM_212524.1Abhd5–––– 1.94 DNA(cytosine-5-)-methyltransferase3alpha NM_001003957.1Dnmt3a?1.52?0.91?1.39?0.74?1.99 Serine(or cysteine)peptidase inhibitor,clade B,member6a NM_199085.1Serpinb6a?0.54?0.65?0.69?0.48?1.68 Hematopoietically expressed homeobox NM_024385.1Hhex–?1.71?1.06?0.57–

The respective GenBank number and the expression level at each time point of exposure is mentioned

Fig.2Heatmap indicating tem-

poral gene expression patterns.

The22common genes between

all the five hypoxia exposure

time points(0.5,1,3,6,and

12h)were depicted along with

symbol and name.The expres-

sion levels were shown in the

bar(red indicates upregulation,

green indicates downregulation,

and black indicates no change).

Comparison between groups of

samples was done with the t test

differential expression algorithm

(detection p value<0.05).The

gene symbol and name were

mentioned

exposure(6.2-fold,p<0.01)as compared to normobaric control rats after which a steady expression level was observed till12h(Fig.6b).Similarly,a significant increase in the expression of HO-1was observed only after6h(5.8-fold,p<0.01)which was further increased after12h(8.0 fold,p<0.001)of exposure(Fig.6b).These results along with IPA analysis and qRT-PCR results suggest that the NRF2-antioxidant responsive element(ARE)pathway is significantly upregulated in response to short-term chronic hypobaric hypoxia in rat brain.

Discussion

Hypobaric hypoxia induces a number of neurological effects on the brain and has a high social and economic impact.As increasing number of people visiting high altitude for work, leisure,and adventure,an understanding of altitude-induced cerebral adaptation,malfunction,management,and treat-ments are becoming increasingly important(Wilson et al. 2009).In an attempt to understand the brain response to hypobaric/environmental hypoxia,we conducted a system-atic expression profiling to identify the genes and molecular events associated with high altitude-exposure.We observed up-or downregulation of108,286,1,020,1,800,and3,740genes(p<0.05)after0.5,1,3,6,and12h of hypoxia exposure respectively(Fig.1a).Higher numbers of upregu-lated genes were detected at0.5,1,3,and6h,while higher numbers of downregulated genes were detected at12h of exposure(Fig.1a).Previous gene expression studies monitoring the brain response to environmental hypoxia have reported regulation of lesser number of genes (Bernaudin et al.2002a;Ganfornina et al.2005;Gustavsson et al.2007;Zhou et al.2008).This could be attributed either by the region specific gene expression like carotid body (Ganfornina et al.2005),cerebral cortex(Gustavsson et al. 2007;Zhou et al.2008)and hippocampus(Zhou et al.2008) or the lower number of analyzed genes(Bernaudin et al. 2002a).In this regard,our whole brain approach combined with temporal monitoring of gene expression,resulting in higher number of identified differential genes represents the most robust gene expression study for environmental hypoxia till date.

Cellular adaptation to hypoxia involves a pronounced repression of energy consuming protein synthesis,which occurs initially at the levels of translation and later extends to the level of transcription as well(Pettersen et al.1986; Hochachka et al.1996;Hochachka and Lutz2001).At the same time,altered expression of many genes and proteins promote metabolic rearrangements including enhanced oxy-

Table2The significant gene ontologies(GO)altered by hypobaric hypoxia in rat brain

Ontology Time of exposure

0.5h1h3h6h12h

GO:0065007:biological regulation 2.94E-04 1.82E-05 2.72E-12 1.12E-17 4.22E-29 GO:0044238:primary metabolic process–– 2.22E-07 2.58E-15 1.25E-23 GO:0008152:metabolic process–– 4.21E-07 1.42E-13 1.51E-23 GO:0050789:regulation of biological process0 6.22E-04 3.16E-09 4.89E-13 2.72E-23 GO:0016043:cellular component organization and biogenesis–– 1.58E-04 1.80E-11 4.08E-23 GO:0044237:cellular metabolic process0.02– 1.11E-08 6.14E-14 6.65E-22 GO:0032502:developmental process9.31E-04 1.06E-07 4.90E-117.55E-16 4.75E-15 GO:0050794:regulation of cellular process0.010.003 1.04E-07 4.09E-10 4.84E-15 GO:0043170:macromolecule metabolic process––7.53E-06 1.25E-08 3.64E-14 GO:0051649:establishment of cellular localization––0.026 5.77E-048.49E-13 GO:0051641:cellular localization––0.031 5.77E-04 3.06E-12 GO:0048519:negative regulation of biological process0.01 4.73E-05 2.10E-07 1.43E-078.66E-12 GO:0051179:localization0.02–0.001 2.73E-06 2.00E-11 GO:0016192:vesicle-mediated transport–––0.002 2.27E-11 GO:0030154:cell differentiation 4.23E-04 3.27E-06 5.48E-089.77E-11 2.33E-11 GO:0048869:cellular developmental process 4.23E-04 3.27E-06 5.48E-089.77E-11 2.33E-11 GO:0048523:negative regulation of cellular process0.017.51E-05 6.56E-07 1.29E-07 1.99E-10 GO:0006810:transport––0.0130.001 2.77E-10 GO:0048468:cell development0.03 3.84E-04 6.22E-06 5.63E-11 4.23E-10 GO:0045045:secretory pathway–––0.0029.23E-10

Top20most significantly regulated ontologies(sorted with p value)were mentioned along with exposure time and p value

gen delivery to hypoxic tissues essential for adaptation and cell survival(Seta and Millhorn2004;Liu et al.2006; Wilson et al.2009).In this study several hypoxia-responsive genes involved in angiogenesis(EGFL7,PGF,ANGPT2, VEGF A),vasodialtion(NOS3),metabolism(PDK4,HKII, PROM1),apoptosis(TIMP3,BNIP3),inflammation (TNFRSF12A,HP,PTGS2),antioxidant response(MT1A, HO-1,DDIT4),and transcription factors(ATF3,NR4A3) were found to be temporally regulated(Table1).The altered expression and contribution of these genes to hypoxic response has been reported for organs including heart(Fan et al.2005;Iacobas et al.2008),liver(Dolt et al.2007;Baze et al.2010),brain(Bernaudin et al.2002a;Zhou et al.2008), lungs(Wu et al.2008;Tada et al.2008),as well as various cells(Mense et al.2006;Polotosky et al.2010).Moreover, genes for angiogenesis and metabolism(Table1)were also found to be differentially regulated during normobaric hypoxia(Bernaudin et al.2002b),ischemia(Hedtjarn et al. 2004),and preconditioning(Stenzel-Poore et al.2003; Carmel et al.2004;Dhodda et al.2004;Gidday2006)in brain.Similarly,genes for antioxidant response(Table1) were reported to be differentially regulated during intermit-tent and sustained hypoxia(Polotosky et al.2010),normo-baric,and hyperbaric hyperoxia(Chen et al.2009a,b

;

Fig.3Analysis of hypobaric hypoxia(12h)regulated network and canonical pathway in brain.Differentially expressed genes from analysis of12-h hypoxia brain sample were analyzed by Ingenuity Pathway Analysis(IPA).a The significant network1(p<0.05) detected by IPA is depicted.The central molecules in the network are FOS and NFE2L2(NRF2).b NRF2-mediated oxidative stress response canonical pathway detected after12h of exposure(p<0.05). Colored genes(red and green)were identified by microarray analysis as differentially upregulated or downregulated in brain samples compared with control.Other uncolored nodal genes are directly or indirectly associated with the differentially expressed genes but were not found to be significantly regulated by hypoxia.Genes are linked by their sub-cellular location(cytoplasm and nucleus).Node(gene) and edge(gene relationship)symbols were described in the bottom part of the figure

Fig.3(continued)

Godman et al.2010)along with ischemic preconditioning (Stenzel-Poore et al.2003;Carmel et al.2004;Dhodda et al. 2004).Our present findings along with these previous studies suggest a tissue-independent overlapping gene signature profile in response to varying oxygen concentra-tion.

Along with hypoxia-responsive genes,many neuroprotec-tive genes like ADM,SGK1,STC1,tP A,ALOX15,and ARC (Table1)were found to be differentially regulated in brain. Adrenomedullin(AM),a neuroprotective peptide is expressed throughout the whole brain(Serrano et al.2000)and upregulated in the cortex region of adult rat brain during acute hypobaric hypoxia(Serrano et al.2008).Recently, Fernandez et al.(2008)have reported that mice with no brain AM have impaired motor coordination,anxiety and were less resistant to hypobaric hypoxia than wild-type mice.The upregulation of serine/threonine protein kinase Serum-and glucocorticoid-induced kinase-1(SGK1)has various function-al roles like facilitation of learning and memory formation (Tsai et al.2002;Chao et al.2007),consolidation of long-term memory(V on Hertzen and Giese2006)and in neuronal plasticity.Stanniocalcin(STC),a glycoprotein hormone plays an important role in maintaining and guarding the integrity of terminally differentiated neuronal cells challenged by ischemia and calcium-mediated cell death(Zhang et al.2000).Tissue-type plasminogen activator(tP A/PLAT)is a highly specific serine proteinase expressed within the central nervous system where it has been associated with learning,synaptic plasticity and regulation of the permeability of the neurovascular unit (Yepes et al.2009).Similarly,the genes impairing neuro-protection like activity-regulated cytoskeletal-associated pro-tein(ARC)/Arg3.1(activity-regulated gene 3.1protein homolog)(Guzowski et al.2000)and12/15-lipoxygenase (ALOX15)(Jin et al.2008)were downregulated during short-term chronic hypoxia(Table1).

Our results also confirm that short-term chronic hypobaric hypoxia altered biological processes and signaling pathways depending on the duration of hypoxia stress(Table2).In response to reduced oxygen supply,rearrangement of meta-bolic pathways has been observed(Hochachka et al.1996; Lopez-Barneo et al.2001),which essentially indicates activation of adaptive responses.The signaling pathways like PI3K/AKT and NF-κB signaling were detected form0.5h of hypoxia exposure demonstrating activation of the survival and proliferation pathways at the onset of the hypoxia exposure (Supplementary material2).The PI3K/AKT survival signaling pathway is regulated by oxidative stress(Wang et al.2000) and activates the nuclear translocation of NF-κB-dependant prosurvival genes(Kane et al.1999).Hypoxia as well as hypoxic preconditioning activates AKT signaling pathway exerting neuroprotection(Alvarez-Tejado et al.2001;Zhang et al.2007;Yin et al.2007).Similarly,activation of NF-κB during cerebral ischemia has been reported to promote proapoptotic as well as antiapoptotic mechanisms(Schneider et al.1999;Irving et al.2000)and plays a key role in the regulation of cellular responses to oxidative stress(Denk et al. 2000).With the increase in exposure time(1–3h)pathways like glycolysis/gluconeogenesis and VEGF signaling were activated(Table3;Supplementary material4)to ensure efficient A TP production by alternate pathways(Hochachka et al.1996;Lutz and Nilsson1997;Hochachka and Lutz 2001)and increased oxygen availability(Marti and Risau 1998).Moreover,activation of VEGF confers neuroprotection after focal cerebral ischemia by PI3K/AKT pathway(Kilic et

Table3The significant canonical pathways altered by hypobaric hypoxia exposure in rat brain

0.5h1h3h6h12h

IL-10signaling Dendritic cell maturation Notch signaling Notch signaling NRF2-mediated oxidative

stress response

Tight junction signaling VDR/RXR activation NRF2-mediated oxidative

stress response MIF regulation of innate

immunity

Antigen presentation

pathway

Arachidonic acid metabolism Nicotinate and

nicotinamide

metabolism

Regulation of actin-based

Motility by Rho

Semaphorin signaling in

neurons

Semaphorin signaling in

neurons

PPARa/RXRa activation Pantothenate and CoA

biosynthesis

IL-8signaling HIF1αsignaling Mitochondrial dysfunction

IL-6signaling Coagulation system Semaphorin signaling in

neurons Nicotinate and

nicotinamide

metabolism

Synthesis and degradation

of ketone bodies

p38MAPK signaling Semaphorin signaling in

neurons VEGF signaling IL-6signaling D-glutamine and D-

glutamate signaling

NRF2-mediated oxidative stress response IGF-1signaling Valine,leucine and

isoleucine degradation

VEGF signaling Hutington’s disease

signaling

4-1BB signaling in T lymphocytes Leukocyte extravazation

signaling

Circadian rhythm

signaling

Circadian rhythm

signaling

Valine,leucine and

isoleucine degradation

The top8significant canonical pathways at each time point were mentioned in order of their p value

A. LCN2

24681012

14Expo s ure time (h)

m R N A e x p r e s s i o n

B. SGK1

0.511.522.5

Expo s ure time (h)

m R N A e x p r e s s i o n

C. ADM

0.511.522.533.5

4Expo s ure time (h)

m R N A e x p r e s s i o n

D. VEGF

0.511.522.53

3.5Expo s ure time (h)

m R N A e x p r e s s i o n

G. SLC16A3

0.511.522.533.5

4Expo s ure time (h)

m R N A e x p r e s s i o n

H. EGFL7

0.511.522.5Expo s ure time (h)

m R N A e x p r e s s i o n

E. HO-1

0.511.522.53

3.5Expo s ure time (h)

m R N A e x p r e s s i o n

F. NOS3

0.511.522.53

3.5Expo s ure time (h)

m R N A e x p r e s s i o n

Fig.4Verification of microarray expression with real-time PCR for differentially regulated gene transcripts.Real-time PCR was per-formed with TaqMan probes with three biological replicates for each time point (0.5,1,3,6,and 12h hypobaric hypoxia exposure,25,000ft).All the samples were run in triplicate (mean ±SE)with endogenous control probes.The expression values were depicted in log 2scale.a LCN2,b SGK1,c ADM ,d VEGF A ,e HO-1,f NOS3,g SLC16A3,h EGFL7,i ALOX15,j EEF2K ,k EPHX2,l PROM1

al.2006).With 6h of hypoxia stress,activation of neuro-protective ERK/MAPK signaling pathway was observed.Both hypoxia and hypoxia-induced ischemic tolerance acti-vates ERK 1/2signaling pathway providing neuroprotection (Fahlman et al.2002;Fang et al.2010).Moreover,activation of ERK pathway also confers neuroprotection during hypo-baric hypoxia (Barhwal et al.2008)and ischemia (Kilic et al.2005;Shioda et al.2009).The longer exposure duration (12h)activated mitochondrial dysfunction along with NRF2-mediated oxidative stress response pathways (Table 3).The sustained oxidative stress during hypoxia leads to mitochondria-permeability transition,release of cytochrome c and mitochondrial dysfunction causing cell death (Calabrese et al.2005;Douglas et al.2010).Hence,activation of mitochondrial dysfunction at 12h of hypoxic exposure indicates activation of pro-apoptotic mechanisms.This was further supported by the major functions like necrosis of cortical neurons,cell death of neurons,injury of nervous tissue,cell death of cortical neurons,cell death of cerebral cortex cells,degeneration of neurons,injury of spinal cord,and injury of axons of molecular networks 1,2,and 4of 12-h exposure (Supplementary Material 4).

Irrespective of exposure period,NRF2-mediated oxidative stress response pathway and genes were found to be significantly regulated in brain (Table 3,Fig.3b ).The transcription factor NRF2is the guardian of redox homeosta-sis and regulates a coordinated induction of a battery of cytoprotective,antioxidant,and anti-inflammatory genes in response to oxidative stress and inflammation (Jaiswal 2004;Nguyen et al.2009).Under normal conditions,NRF2undergoes proteasomal degradation in cytoplasm by Kelch ECH associating protein 1(KEAP1).During oxidative stress NRF2is released from KEAP1,translocates to the nucleus and transactivates the expression of several dozen cytopro-tective genes containing cis -acting ARE (Kensler et al.2007;Nguyen et al.2009).Increasing number of studies has suggested that NRF2-ARE activation is a novel neuro-protective pathway that confers resistance to a variety of oxidative stress-related neurodegenerative insults like 6-hydroxydopamine (Jakel et al.2007),kainic acid (Kraft et al.2006),hypobaric hypoxia (Barhwal et al.2009),cerebral ischemia (Shih et al.2005a ;Shah et al.2007),intracerebral hemorrhage (Wang et al.2007;Zhao et al.2007),and hyperoxia toxicity (Chen et al.2009a ,b ).Moreover,NRF2pathway-mediated promotion of neuroprotection by neurite outgrowth-promoting prostaglandins (Satoh et al.2006),sulforaphane (Innamorato et al.2008),and acetyl-l-carnitine (Barhwal et al.2009)also suggests that NRF2is a therapeutic target against neurodegenration (Johnson et al.2008).High altitude exposure causes oxidative stress in brain via generation of reactive oxygen and nitrogen species which can cause damage to lipids,proteins,and DNA (Moller et al.2001;Maiti et al.2006;Hota et al.2007).Brain being exquisitely sensitive to oxygen has evolved a dynamic

I. ALOX15

Exposure time (h)m R N A e x p r e s s i o n

J. EEF

2K

Exposure time (h)m R N A e x p r e s s i o n

K. E

PHX2

Exposure time (h)

m R N A e x p r e s s i o n

L. PROM1

Exposure time (h)

m R N A e x p r e s s i o n

Fig.4(continued)

protective response to altered oxygen concentrations and oxidative stress (Sharp and Bernaudin 2004;Wilson et al.2009).Our expression analysis combined with pathway identification by IPA suggests NRF2-mediated oxidative stress response is the major pathway providing cytoprotection during short-term chronic hypobaric hypoxia (Fig.3a and b ).We also observed upregulation of NRF2downstream targets HO-1,SOD ,TXN ,GST ,GCLC ,and GCLM in response to hypobaric hypoxia (Fig.3b ).Similar whole genome expres-sion studies assessing the effects altered oxygen concentration like intermittent and sustained hypoxia in human arotic endothelial cells (Polotosky et al.2010),hypoxia-ischemia in immature mice brain (Hedtjarn et al.2004),ischemia-reperfusion injury in kidney (Leonard et al.2006),and

normobaric and hyperbaric hyperoxia in rat cortical neurons (Chen et al.2009a ,b )have also identified upregulation of transcription factor NRF2and downstream genes.Validation of mRNA abundance of NRF2by real-time PCR also revealed higher expression levels in various brain regions following 12h of hypoxia (Fig.5).Similarly,expression of HO-1was also found to be temporally induced by hypoxia (Table 1;Fig.4e )and in various brain regions following 12h hypoxia (Fig.5).The induction of HO-1is considered to counteract oxidative damage,confer cytoprotection,and promote adaptive survival (Ryter et al.2006;Gozzelino et al.2010).In corroboration,our western blot results also demonstrate temporal activation of NRF2and HO-1proteins in response to short-term chronic hypobaric hypoxia (Fig.6).The maximum expression of NRF2was observed after 3h of exposure while the expression of HO-1was observed after 6h of exposure supporting the stabilization and nuclear transport of NRF2followed by transcriptional activation of HO-1(Jaiswal 2004;Kensler et al.2007;Nguyen et al.2009).The activation of NRF2/HO-1pathway provides neuroprotection against oxidative stress (Satoh et al.2006

;

2000

40006000800010000120001400016000C

0.5 h

1 h

3 h

6 h

12 h

Exposure time (h)

R e l a t i v e i n t e n s i t y (a r b i t r a r y u n i t s ) a f t e r n o r m a l i z a t i o n

β

-ACTIN

NR F 2

HO-1

C 0.5h 1h 3h 6h 12h

a

b

Fig.6Analysis of NRF2and HO-1expression in rat brain after 0.5,1,3,6,and 12h of hypobaric hypoxia exposure (25,000ft)by western blot analysis.a Representative blot of NRF2and HO-1temporal expression in brain of hypobaric hypoxia exposed rats along with normobaric controls.b Average NRF2and HO-1expression in normobaric control and hypobaric hypoxia exposed brain depicted graphically after normalization to β-action expression.Student ’s t test was performed and p <0.05was considered statistically significant (*p <0.05,**p <0.01,and ***p <0.001with respect to normobaric control)

123456

7m R N A e x p r e s s i o n

0.511.522.533.54

4.5m R N A e x p r e s s i o n

0.511.522.533.5

4LCN2

SGK1

ADM

VEGFA

NRF2

HO-1

NOS3SLC16A3

EGFL7m R N A e x p r e s s i o n

Fig.5Expression analysis of identified differential genes in brain regions (amygdala,hippocampus,cortex,and cerebellum)exposed to 12h of hypobaric hypoxia (25,000ft).Real-time PCR was performed with TaqMan probes for LCN2,SGK1,ADM ,VEGF A ,NRF2,HO-1,NOS3,SLC16A3,and EGFL7from three biological replicates (mean±SE).All the reactions were run in triplicate with endogenous control.The expression levels are in log 2scale

Innamorato et al.2008;Espada et al.2010)and promotes neuronal survival(V argas et al.2005).Altogether,our study suggests that upregulation of transcription factor NRF2and activation of NRF2-ARE pathway as an intrinsic defense mechanism of brain against short-term chronic hypobaric hypoxia-induced oxidative stress.

The intensity and duration of stress along with the individual response determines the final outcome.In the present study,activation of signaling pathways(NF-κB, PI3K/AKT,and ERK/MAPK)along with protective genes during short-term chronic hypobaric hypoxia(Table1)may represent rapid activation of antiapoptotic pathways (Guyton et al.1996;Milton et al.2008).But the delayed and sustained activation of both NF-κB and ERK/MAPK has been reported in contributing to apoptosis in response to severe oxidant injury and brain ischemia(Martindale and Holbrook2002;Vartiainen et al.2003;Song et al.2008). Similarly,the activation of mitochondrial dysfunction path-way after12h of exposure also suggests a diminished activity of mitochondrial function-stabilizing pathways and proteins(Kilic et al.2005)during longer hypoxic exposure. Though we have not measured the expression levels of these pro-and anti-apoptotic molecules in longer hypoxia expo-sure,such studies may substantiate the current findings.In parallel,hypoxia exposure also induced pro-inflammatory cytokines like IL-6,IL-8as well as anti-inflammatory cytokine IL-10signaling pathways(Table3).During hypoxia,increased ROS production leads to activation of many proinflammatory cytokines by various mechanisms (Yan et al.1995;Tamm et al.1998),whereas IL-10treatment provides neuroprotection to hippocampus after ischemia (Dietrich et al.1999)and promotes angiogenesis in ischemic tissue(Silvestre et al.2001).These cumulative results suggest simultaneous activation of neuroprotective,antioxi-dant,anti-and proinflammatory genes during short-term chronic hypobaric hypoxia.

In summary,the global expression analysis of rat brain under short-term chronic hypobaric hypoxia followed by IPA analysis has provided a detailed insight of hypoxia-regulated brain transcriptome.The whole brain approach with temporal monitoring of gene expression has enabled us in(a)identification of hypoxia-responsive genes and expression patterns(b)deciphering the sequence of signal-ing events underlying hypoxia,(c)depicting the intrinsic defense mechanisms,and(d)revealing hypoxia-regulated novel genes in brain.Further,our studies have also identified and validated of NRF2-mediated oxidative stress response pathway as one of the major intrinsic antioxidant response of the brain in response to short-term hypobaric hypoxia.The results from this study will open the avenues for therapeutic interventions targeting the NRF2pathway for the early amelioration of hypobaric–hypoxia-induced neuropathologies.Acknowledgment This study was supported by Defence Institute of Physiology and Allied Sciences Grant TC/297/TASK-122(NKS)/ DIPAS/2007.MS is a recipient of Defence Research and Development Organisation(DRDO)Senior Research Fellowship(SRF).The technical support provided by Dr.Dhananjay Shukla is greatly appreciated.The authors declare no conflict of interest. References

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