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Physiol. Genomics 32: 370-379, 2008. First published December 4, 2007; doi:10.1152/physiolgenomics.00147.2007
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Received 10 July 2007; accepted in final form 3 December 2007.
Physiological Genomics 32:370-379 (2008)
1094-8341/08 $8.00 © 2008 American Physiological Society

Gene expression in mouse brain following chronic hypoxia: role of sarcospan in glial cell death

Dan Zhou 1,*, Jiyi Wang 1,*, Matthew A. Zapala 2, Jin Xue 1, Nicholas J. Schork 2 and Gabriel G. Haddad 1,3

1 Department of Pediatrics (Section of Respiratory Medicine) and Neuroscience, University of California San Diego, La Jolla
2 Department of Psychiatry, University of California San Diego, La Jolla
3 The Rady Children's Hospital-San Diego, San Diego, California


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypoxia is a hallmark of respiratory, neurological, or hematological diseases as well as life at high altitude. For example, chronic constant hypoxia (CCH) occurs in chronic lung diseases or at high altitude, whereas chronic intermittent hypoxia (CIH) occurs in diseases such as sleep apnea or sickle cell disease. Despite the fact that such conditions are frequent, the cellular and molecular mechanisms underlying the effect of hypoxia, whether constant or intermittent, are not well understood. In this study, we first determined the effect of CCH and CIH on global gene expression in different regions of mouse brain using microarrays and then investigated the biological role of genes of interest. We found that: 1) in the cortical region, the expression level of 80 genes was significantly altered by CIH (16 up- and 64 downregulated), and this number increased to 137 genes following CCH (34 up- and 103 downregulated); 2) a similar number of gene alterations was identified in the hippocampal area, and the majority of the changes in this region were upregulations; 3) two genes (Sspn and Ttc27) were downregulated in both brain regions and following both treatments; and 4) RNA interference-mediated knockdown of Sspn increased cell death in hypoxia in a cell culture system. We conclude that CIH or CCH induced significant and distinguishable alterations in gene expression in cortex and hippocampus and that Sspn seems to play a critical role in inducing cell death under hypoxic conditions.

expression profile; central nervous system; oxygen deprivation; development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
IT IS WELL KNOWN THAT ENVIRONMENTAL factors that modulate normal growth conditions at an early postnatal age can have a long-lasting effect on brain function and structure. Oxygen deprivation can occur in many childhood diseases such as obstructive sleep apnea (OSA) (5), brochopulmonary dysplasia (BPD) (28), congenital heart disease (CHD), asthma, epilepsy, and sickle cell disease (SCD). The affected infants often have long-lasting pathological outcomes accompanied with cognitive and/or behavioral disturbances (4, 11, 13, 19, 21, 29). The molecular mechanisms underlying the effects of hypoxia on postnatal brain development are now still largely unknown.

In rodents, the rapid phase of cortical maturation and hippocampal development during the first few postnatal weeks corresponds to the first 2–3 postnatal years of rapid cortical and hippocampal development in humans (2). Using the mouse model, previous studies in our laboratory and those of others have shown that O2 deprivation can affect brain development in a major way. For example, an increase of capillary density, a decrease in myelination and a decrease of N-acetylaspartate-to-creatinine ratios are some of the major findings (9, 12). To further investigate the mechanisms underlying hypoxic injury or adaptation in the developing brain, we identified in this work the hypoxia-induced alterations in gene expression and biological networks in the neocortical and hippocampal regions following long-term postnatal hypoxic treatment. We chose these two important central nervous system (CNS) regions because they are known to be vulnerable to hypoxia (20). Two long-term hypoxia treatment paradigms were applied separately to mimic the hypoxic conditions that can take place in different environmental or health conditions. One paradigm is a chronic intermittent hypoxia (CIH) that mimics conditions such as OSA. The other paradigm is a chronic consistent hypoxia (CCH) that mimics conditions such as BPD, SCD, and being at high altitude.

Early postnatal brain development includes growth of axons, formation of specific synaptic connections, and the fast proliferation of glial cells. Therefore, the postnatal development of the brain should no longer be considered solely as the development of neuronal network contacts, but also formation of the integrated circuitries of interactive neurons and glial cells. Indeed, it has been shown that chronic neonatal hypoxia led to decrease of volume and cell number in the cerebral cortex, especially a reduction of glial cells in white matter (3, 25). A better understanding of glial cell biology, especially their responses to chronic hypoxia, can offer potential hints for developing novel therapeutic strategies to treat hypoxia-induced neurological disorders. In the current study, we determined the gene expression profiles in the neocortex and hippocampus of developing mouse brain following CCH or CIH treatments. We found that the expression of sarcospan (Sspn) gene was significantly downregulated in both brain regions following either CCH or CIH treatment. The role of Sspn in cell survival under hypoxia was further determined in cultured glioma cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals, hypoxia treatment, and tissue preparation.
CD-1 mice (Charles River, MA) were utilized for all experiments. This mouse strain has been extensively evaluated in our laboratory (9, 10, 12). All litters were culled to eight pups each. On the second day after birth (P2), litters and their mothers were placed in Plexiglas chambers. In CCH experiments, 11% of O2 concentration was applied continuously. In CIH experiments, O2 concentration was changed in cycles between 21% for 4 min and 11% for another 4 min throughout 24 h/day (10). Control litters were housed in identical chambers and exposed to room air. Animals were housed under 12-h light/dark cycles with free access to food and water. After 2 wk of treatment, the mice were decapitated after halothane anesthesia, and the brains were rapidly removed. Four brain regions (cortex, hippocampus, brain stem-diencephalon, and cerebellum) were quickly separated on ice, flash-frozen in liquid nitrogen, and stored at –80°C till use. All animal protocols were approved by the Animal Care Committee of Albert Einstein College of Medicine of Yeshiva University, where the animal treatment and sample collection were performed before our lab moved to the University of California, San Diego.

Total RNA purification and cRNA labeling.
Total RNA was extracted from frozen cortical and hippocampal tissues of three individual mice for each treatment (normoxia, CIH, and CCH) using RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The RNA quality was determined by Agilent 2100 bioanalyzer (Agilent Technologies). Total RNA (300 ng) was used to create a biotin-labeled complementary RNA (cRNA) by the Illumina RNA amplification kit (Ambion) according to the manufacturer's instructions.

Microarray hybridization, image acquisition, and data analyses.
The Sentrix mouse-6 expression bead chips (Illumina, San Diego, CA) were used to determine differences in gene expression (14). Each bead chip contains six whole-genome gene expression arrays, allowing six samples to be hybridized to a single chip and with >47,000 probes to query expression profiles of the mouse genome. Hybridizations were carried out using the Illumina gene expression system according to the manufacturer's instructions. In brief, biotin-labeled cRNA (1.5 µg) was added to the array and incubated for 16–20 h at 55°C. The bound biotin-labeled cRNA was then stained with streptavidin-Cy3. Three chips were processed. Each chip contains six individual arrays that can measure the gene expression levels of six individual samples. We processed a group of six samples with each chip that included one control-cortical sample, one CIH-cortical sample, one CCH-cortical sample, one control-hippocampal sample, one CIH-hippocampal sample, and one CCH-hippocampal sample. Thus, three individual measurements were performed for each treatment in each brain region. After hybridization, the microarray chip was washed, dried, and scanned by the Illumina BeadArray Reader, the absolute intensity of each probe on the image was generated with BeadStudio software (Illumina). The difference in expression level was calculated by comparison between the group of treated samples and the group of controls from each chip after cubic spline normalization. The changes were considered significant when the fold changes were >1.50, and the differential scores (i.e., adjusted P values) were >20 (for upregulations) or –20 (for downregulations), which is equivalent to P < 0.01 according to the manufacturer's instruction. The data set from microarray analyses can be traced under the Gene Expression Omnibus (GEO) series access number GSE8262 in the National Center for Biotechnology Information GEO database (http://www.ncbi.nlm.nih.gov/geo).

Ingenuity pathways analysis.
Microarray data were further analyzed through the use of Ingenuity Pathways Analysis (Ingenuity Systems, www.ingenuity.com). Ingenuity Pathway Analysis is a web-based application that enables the discovery, visualization, and exploration of therapeutically relevant gene networks. It was used to generate specific biological networks in our gene-expression array data sets. Four data sets containing lists of differentially expressed genes in either cortical or hippocampal region following CIH or CCH treatment were uploaded to Ingenuity as individual tab-delimited text files. Each list was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base via the Refseq number. These genes, called Focus Genes, were then used as the starting point to generate biological networks. To build the relevant networks, the application queried the Ingenuity Pathways Knowledge Base for interactions between Focus Genes and all other gene objects in the knowledge base and generated a set of networks with a network size of ~35 genes. A significant score was computed for each network according to the size of the network and the fit of the uploaded set of significant genes. This score is based on a right-tailed Fisher's exact test identifying networks that have significantly more focus genes than expected by chance. The biological functions were calculated and assigned to each network by using the Ingenuity Pathways Knowledge Base. The biological functions assigned to each network are ranked according to the significance of that biological function to the network. Again, a right-tailed Fisher's exact test is used to calculate a P value determining how likely it is that genes from that network belong to a specific biological function.

Culture of LN-229 cells.
LN-229 cells (ATCC number: CRL-2611) were plated in 75-cm2 flasks. DMEM was supplemented with 10% FBS and 1% penicillin-streptomycin. The cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 and 21% O2. The medium was changed twice a week.

Cells were dissociated with 0.25% trypsin/EDTA at 100% confluence and then diluted in 48-well dish so that such dishes contained the appropriate number of cells and give 30–50% confluence within 24 h after plating. After 1 day of culture under normoxia, one of the 48-well dishes was transferred into the hypoxic incubator with 1.5% O2, 5% CO2 at 37 °C. Cell proliferation and cell death were analyzed after 3 days or 5 days of treatment under either normoxia or hypoxia.

RNA interference reverse transfection for Sspn knockdown.
Sspn knockdown in LN-229 cells was performed with Sspn Stealth Select RNA interference (RNAi; Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Briefly, 12 pmol RNAi and 40 µl Opti-MEM Medium without serum were mixed in the well of 48-well plate. Then 0.6 µl Lipofectamine RNAiMAX was added and incubated for 10–20 min at room temperature. Cells were diluted in 200 µl of medium without antibiotics to give 30–50% confluence within 24 h after plating. The diluted cells of 200 µl were added into the well with RNAi and Lipofectamine RNAiMAX. The cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 1 day. Then one of the 48-well dishes was transferred into a CCH incubator with 1.5% O2 (balanced with N2), 5% CO2 at 37°C. After 3 days or 5 days treatment under normoxia and hypoxia, cells were taken out from the incubator for cell proliferation or LDH assay.

KIF11 Stealth Select RNAi was used as positive control to assess transfection efficiency. Cells in which KIF11 was knocked down arrested during mitosis, and exhibited a "rounded-up" morphology in adherent cells after 24–30 h. We assessed transfection efficiency by determining the number of arrested cells (>50%).

Semiquantitative RT-PCR assay.
Semiquantitative RT-PCR was also used to confirm the mRNA levels of five selected genes identified by microarray as well as the level of Sspn mRNA following RNAi transfection. All specific primers were designed by the software Primer 3 (23) and synthesized at ValueGene (San Diego, CA) (Supplementary Table S2).1 The validation of the microarray results was done by determining the changes in the same total RNA samples used in the microarray experiments. Briefly, first-strand cDNA was synthesized from 1 µg of total RNA using SuperScriptII reverse transcriptase and Oligo-(dT) primer in a 20 µl reaction volume according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). The cDNA (100 ng) was used for each PCR reaction. The number of PCR cycles used for each primer is shown in Table 1. Mouse β-actin was used as internal control and the final result represents the fold change of treated samples over controls. The experiment was done in triplicate.


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  		Table 1. List of genes altered by both CIH and CCH in cortex

 
To confirm the knockdown of Sspn mRNA following RNAi transfection, total RNAs were extracted from the LN-229 cells with or without RNAi transfection using the RNeasy kit (Qiagen, Valencia, CA). The level of Sspn mRNA was analyzed using the specific primers (forward: 5'-TGCTCAAAGTTGCGAGAGAA-3' and reverse: 5'-AAGCATTTGGCAAGTGATCC-3'), human β-actin gene was used as internal control (forward primer: 5'-GGACTTCGAGCAAGAGATGG-3'; reverse primer: 5'-CACCTTCACCGTTCCAGTTT-3'). The isolated total RNA was treated with DNaseI (Roche, Indianapolis, IN) to remove genomic DNA. First strand cDNA was synthesized from 50 ng of total RNA using SuperScriptII reverse transcriptase and Oligo-(dT) primer in a 20 µl of volume reaction (Invitrogen). The cDNA (5 ng) was used for PCR reaction, and the number of PCR cycles used was 40. The final result represents the fold change of treated samples over normoxic control. The experiment was done in triplicate.

Cell proliferation assay.
Cell proliferation assays were performed using an in situ cell proliferation kit (Roche), according to the manufacturer's instructions. In brief, cells were incubated with 5-bromo-2'-deoxyuridine (BrdU) for 60 min at 37°C. Cells were fixed with fixative solution containing 3 volumes of glycine solution (50 mM, pH 2.0) with 7 volumes of absolute ethanol for 45 min, treated with 4 M HCl at room temperature for 20 min, and incubated with anti-BrdU-FLUOS for 45 min at 37°C. Cells were counterstained with propidium iodide (1 µg/ml) for counting the total cell number. The BrdU-positive cells were counted by randomly scoring 10 fields for each treatment in each independent experiment (n = 3), and the data are presented as the percentage of BrdU-positive cells relative to the total number of cells.

Lactate dehydrogenase assay.
Lactate dehydrogenase (LDH) activity in cells and supernatant was determined using a LDH assay kit (Sigma, St. Louis, MO) according to the manufacturer's instructions. Briefly, the supernatant containing the released LDH from the damaged cells was removed into a microcentrifuge tube. The cells remaining in the plate were lysed to release all intracellular LDH. The LDH released from the damaged cells and from the lysed cells was separately subjected to the LDH assay. In brief, 100 µl of the mixture of LDH-assay substrate, cofactor, and dye solution (1:1:1) was added to each 50 µl of supernatant or 50 µl of cell lysate, and the plate was incubated at room temperature for 30 min, followed by the addition of 15 µl of 1 N HCl to each well. The assay is based on the presence of released, cellular LDH in the culture media. The presence of LDH allows for the conversion of NAD into NADH, which then causes a color reaction of a tetrazolium dye substrate. The colored product of the transformed tetrazolium dye can be measured spectrophotometrically at 490 nm. The absorbance at 490 nm (A490) was measured using a Synergy HT microplate reader (BioTek, Winooski, VT). Duplicate wells were read for each sample, and the experiment was done three times. The percentage of LDH released from the damaged cells was calculated by the following formula:

Formula

Statistical analysis.
The statistical significances of the results from semiquantitative RT-PCR, cell proliferation, and viability assays were calculated by a two-tailed Student's t-test using Microsoft Excel software and expressed as means ± SD. Differences in means were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Overview of gene expression profiles.
Using long-oligo expression arrays, we determined the changes in gene expression in both cortex and hippocampus following either CIH or CCH. In the cortex, the expression levels of 80 transcripts were significantly altered following CIH (16 upregulated and 64 downregulated). This number increased to 137 following CCH (34 upregulated and 103 downregulated) (Supplementary Table S1). In the hippocampus, however, 71 transcripts were significantly altered following CIH (57 upregulated and 14 downregulated), and 80 transcripts were differentially expressed following CCH (69 upregulated and 11 downregulated) (Supplementary Table S1). In contrast to the cortex, most of the significant changes in the hippocampal region were upregulations. Out of all the altered transcripts, 22 transcripts responded to both CIH and CCH in the cortex (2 upregulated and 19 downregulated) (Table 1). In the hippocampal region, 11 transcripts were found to be significantly altered following both CIH and CCH, and most of them were upregulated (Table 2). Interestingly, there were more transcripts that were significantly altered following CCH compared with CIH. For example, following CIH, 26 transcripts were altered in both cortical and hippocampal regions (12 upregulated and 14 downregulated, Table 3), whereas, following CCH, 37 transcripts changed their expression in both cortical and hippocampal regions (26 upregulated and 11 downregulated, Table 4). Only two genes were found to be altered (i.e., downregulated) in both brain regions following both treatments. One of them encodes Sspn and the other one encodes for tetratricopeptide repeat domain 27 (Ttc27).


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  		Table 2. List of genes altered by both CIH and CCH in hippocampus

 

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  		Table 3. List of genes altered in both cortex and hippocampus following CIH

 

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  		Table 4. List of genes altered in both cortex and hippocampus following CCH

 
The data from the microarrays were evaluated using semiquantitative RT-PCR. As presented in Fig. 1, the data from the microarrays and semiquantitative real-time PCR were qualitatively similar.


Figure 1
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  		Fig. 1. Validation microarray results by semiquantitative RT-PCR. Microarray results of 5 selected genes were validated by semiquantitative RT-PCR. The changes were consistent between microarray and semiquantitative RT-PCR measurement (correlation coefficient = 0.84). The ratio of each gene from semiquantitative RT-PCR was normalized to β-actin, and the values are means ± SD of values from triplicate experiments. Sspn, sarcospan.

 
Distinct gene expression following CIH or CCH.
The significantly altered genes were categorized based upon the magnitude of change. We found that: 1) most genes in the cortex were downregulated following CIH (~80%) or CCH (~75%). Although the majority of these changes were less than twofold following either CIH or CCH (Fig. 2, B and C), a number of genes changed by more than 3- and some by even 10-fold. For example, in the CIH-treated cortical region, Bat5 and an MHC gene, H2-L, were downregulated by over fourfold. On the other hand, a relatively smaller number of genes were upregulated following CIH (~20% of total significant changes) and CCH (~25% of total significant changes) in the cortex, and the most upregulated gene was H2-D1 (>5-fold) after CIH (Fig. 2, B and C; Supplementary Table S1). 2) In contrast to the cortex, most of the altered genes were significantly upregulated following either CIH (~80%) or CCH (~85%) treatment (Fig. 2, D and E).


Figure 2
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  		Fig. 2. Summary of gene expression profiles in developing mouse brain following chronic intermittent hypoxia (CCH) or chronic constant hypoxia (CIH). A: Venn diagram illustrating the number of significantly altered genes and the overlap of genes between different treatments and regions. B: gene expression profile of cortex following CIH. C: gene expression profile of cortex following CCH. D: gene expression profile of hippocampus following CIH. E: gene expression profile of hippocampus following CCH.

 
Distinct alterations in biological networks following CIH or CCH.
The significant changes in both brain regions following CIH or CCH were further categorized into functional networks, based upon interactions between the significantly altered genes, using Ingenuity pathways analysis. Distinct functional networks were identified in the brain regions with different treatments (Table 5, Supplementary Tables S3–S6). In the cortical region, CIH induced alterations that were categorized into three distinct networks that regulate CNS function, cell death, and various neurological diseases or conditions. CCH induced alterations in five networks that regulate cell signaling and CNS development and metabolism. On the other hand, in the hippocampal region, CIH induced alterations in the networks that regulate mainly inflammatory/immune responses and cell death. CCH, however, induced changes that are mainly related to cellular development and vascular growth/oxygen transport (Table 5).


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  		Table 5. List of representative significant functional networks identified by Ingenuity Pathways Analysis in cortex or hippocampus following CIH or CCH treatment

 
Sspn downregulation induces cell death.
Sspn is a member of the tetraspan superfamily that has been implicated in cell signaling, such as cell adhesion, migration, and proliferation (15, 17, 30). Interestingly, also, Sspn seems to have a role in ROS generation and scavenging (Fig. 3). In the current study, Sspn is the only characterized gene that was found to be downregulated in all conditions and brain regions, suggesting that Sspn plays an important role during O2 deprivation by affecting processes such as cell proliferation and survival. To test this hypothesis, we studied the effect of Sspn knockdown on cell proliferation or death in a cultured glioma cell line (LN-229) using BrdU incorporation or LDH release assays.


Figure 3
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  		Fig. 3. Graphical representation of Sspn interaction network. The relationship of proteins and small molecules that interact with Sspn was summarized by Network analysis using Ingenuity knowledge database. The network indicates that Sspn directly or indirectly regulates level of ROS in the cell.

 
As shown in Fig. 4, RNAi probes targeting Sspn significantly downregulated the level of Sspn mRNA (Fig. 4A). Following a single administration of BrdU, though, a significant decrease in BrdU incorporation was found in both cells with or without Sspn knockdown under hypoxic condition compared with normoxic condition (19.14 vs. 3.84%, P < 0.01), there was only a small and statistically insignificant decrease of BrdU incorporation between control and Sspn knockdown cells under either normoxia or hypoxia (Fig. 4B).


Figure 4
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  		Fig. 4. Effect of Sspn downregulation on cell proliferation or death. A: RNA interference (RNAi)-Sspn treatment significantly decreased the level of Sspn mRNA in LN-229 cells. B: effect of Sspn downregulation on 5-bromo-2'-deoxyuridine (BrdU) incorporation in LN-229 cells. Although hypoxia treatment significantly reduced BrdU incorporation in LN-229 cells, no significant differences in BrdU incorporation were found between Sspn knocked-down and control cells under either normoxic or hypoxic conditions. C: effect of Sspn downregulation on cell death in LN-229 cells. Viability of controls and RNAi-Sspn-treated cells were measured after 5 days of hypoxic treatment by the release of LDH. RNAi-Sspn treatment significantly increased cell death under normoxic and hypoxic conditions. Values are means ± SD. Statistical significance was calculated by Student's t-test. **P < 0.01 compared with normoxic controls; ++P < 0.01 compared with hypoxic controls; #P < 0.05 compared with normoxic RNAi-Sspn-treated cells.

 
The effect of Sspn downregulation on cell death was determined by LDH release. We found that Sspn knockdown significantly increased LDH release compared with control during normoxia (9.55 vs. 34.37%, P < 0.01) and after 5 days of hypoxia (12.52 vs. 51.07%, P < 0.01) (Fig. 4C). No significant difference in LDH release was found in controls following normoxia or hypoxia treatment. Interestingly, however, Sspn knockdown increased the vulnerability of LN-229 cells to hypoxia. LDH release in Sspn knockdown cells was significantly higher following hypoxia than following normoxia (34.37 vs. 51.07%, P < 0.05) (Fig. 4C). A similar pattern was also detected in Sspn knockdown cells following 3 days of hypoxia (data not show).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Diseases that occur during childhood, such as OSA, BPD, asthma, and SCD, can often have long-term pathological outcomes accompanied by cognitive and/or behavioral disturbances. Since one of the important elements of these diseases or conditions is low oxygenation, we wished to study the effect of hypoxia on the CNS, especially on neocortex and hippocampus, because the disturbances and defects in these children suggested the involvement of these brain regions. To understand and dissect the mechanisms underlying the effects of CIH or CCH on cortical and hippocampal development, genome-wide gene expression profiles were evaluated in the neocortex and hippocampus. This is the first systematic investigation of the expressional response to long-term CCH or CIH in the developing mouse brain in the first few weeks of life. The reason for choosing this exposure time (the first 2 wk) is that this period corresponds to the early developmental stage (months to 2 yr) in humans (2).

Both hypoxia paradigms, CIH and CCH, induced major changes in the developing mouse brain. Of interest, the gene expression response to hypoxia in the developing brain seems to be region and hypoxia paradigm specific. For example, the majority of changes was essentially a downregulation in the neocortex (~80% following CIH and ~75% following CCH) but an upregulation in the hippocampus (~80% following CIH and 85% following CCH), demonstrating that hypoxia induced more suppression on gene expression in the neocortex, but more of an activation in gene expression in the hippocampus. Such dramatic differences demonstrated also that distinct transcriptional regulatory mechanisms were involved in a tissue/cell-specific manner to cope with oxygen deprivation in the brain. Furthermore, pathway analysis showed that CIH and CCH induced different alterations in functional networks. For instance, although CIH and CCH both induced an upregulation of many genes in the hippocampus, the alterations induced by CIH were mainly in the networks that regulate inflammatory and immune responses that might lead to a variety of neurological diseases. In contrast, the alterations induced by CCH were mainly in the networks that contribute to oxygen transport and cellular rearrangement that might lead to an adaptation in the brain (Table 5). These results suggest different impacts of CIH and CCH on the brain. It seems that CCH induced more of an "adaptive response," whereas CIH induced more of a pathological one. Indeed, CCH, but not CIH, induced a coordinated upregulation of aminolevulinic acid synthase 2 (erythroid, Alas2) and globins in both cortical and hippocampal regions, which demonstrated a synchronized upregulation of heme and globin production and hence an increase in oxygen transport capacity that can cope with oxygen deprivation in the brain. On the other hand, glyoxalase 1 (Glo1) was downregulated in both hippocampus and cortex following CIH. As glyoxalase system plays an important role in methylglyoxal detoxification that reduces the possible occurrence of oxidative stress in cells (1, 8, 22), downregulation of this enzyme might enhance the cytotoxicity of methylglyoxal, therefore, causes CNS injury in the developing brain following CIH treatment. In addition, in the current study, most of the altered genes were for the first time related to hypoxia-induced functional disturbances in the brain. For example, 14 significantly altered genes were categorized into a network that contributes to neurological diseases and psychological disorders in the cortex following CIH treatment (Table 5). According to a PubMed search, 10 of them were related to hypoxia responses for the first time in the current study.

The downregulation of Sspn in our work induced significant cell death without a profound effect on cell proliferation in a glioma cell culture (Fig. 4, B and C). Sspn is a member of the tetraspan protein superfamily and is originally identified as one of the essential components of the sarcoglycan-sarcospan-dystroglycan complex. Along with syntrophins, dystrobrevins, and a diverse group of signaling proteins, Sspn is associated intimately with dystrophin at the sarcolemma of skeletal, cardiac, and smooth muscle (6, 7, 26, 31). The current study constitutes the first report demonstrating that Sspn is involved in hypoxia response in the developing brain. Although the exact mechanism remains elusive, the effect of Sspn downregulation on glial cell survival might be related to the destabilization of the dystrophin complex by Sspn knockdown on the cell surface and, hence, the disturbances of cell adhesion and/or signal transduction pathways, or through affecting the level of reactive oxygen species and the activity of superoxide dismutase (Fig. 3) (16, 24).

One question that can be asked is how is the brain or the whole organism trying to survive hypoxia? And, in particular, how does Sspn help the organism by its downregulation, if the effect of its downregulation is to induce cell death in the CNS? The answer to this question could be obtained, at least in part, from our studies and those of others. Note that: a) hypoxia has induced less BrdU incorporation in cells whether wild-type or Sspn knock-down, indicating that hypoxia does decrease the ability of cells to proliferate; b) it seems that in chronic hypoxia, especially in CCH, there is an increase in angiogenesis and an increase in Alas2 and globins, demonstrating an increase in heme and globin production. These would strongly suggest that there is an increase in oxygen carrying capacity; c) surprisingly, Sspn, by its downregulation, induces cell death, and this would indicate that Sspn, under a transcriptional control, is programmed to play an important role in hypoxia; d) it has been demonstrated that hypoxia inhibits cell differentiation and proliferation and increase the number of stem cell-like progenitor cells in CNS (18, 27, 32, 33). Taken together, some of the overarching strategies in hypoxia would be: 1) to increase oxygen carrying capacity and transport, 2) to decrease the number of cells or cell differentiation and proliferation to decrease the overall oxygen demand, and 3) to keep cells in a hypoxia-tolerant state such as for stem cell-like progenitors.

In summary, our results demonstrated that CCH and CIH induced significant alterations in gene expression in the cortical and hippocampal regions of the developing brain. The biological networks and processes responding to CIH and CCH are different in both brain regions with little overlap. Hypoxia induced a more profound downregulation in gene expression in the cortical area but an upregulation in the hippocampus. Such differences indicated that hypoxia evoked predominantly a transcriptional suppression in the cortical region but a transcriptional activation in hippocampus. Furthermore, downregulation of Sspn gene in glial cells significantly affected cell survival under hypoxic conditions, which suggests Sspn may play an important role in mediating hypoxic injury during postnatal brain development.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institute of Child Health and Human Development Grant PO1HD-032573 (to G. G. Haddad) and a Parker B. Francis Foundation fellowship grant to J. Xue.


   FOOTNOTES
 
Address for reprint requests and other correspondence: G. G. Haddad, Dept. of Pediatrics and Neuroscience, Univ. of California, San Diego, 9500 Gilman Drive, MC 0735, La Jolla, CA 92093-0735 (e-mail: ghaddad{at}ucsd.edu).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

* D. Zhou and J. Wang contributed equally to this work. Back

1 The online version of this article contains supplemental material. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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