High-throughput Functional Genomics Identifies Genes That Ameliorate Toxicity Due to Oxidative Stress in Neuronal HT-22 Cells

GFPT2 Protects Cells Against Peroxide*

Jürgen Zitzler{ddagger}, Dieter Link{ddagger}, Rolf Schäfer{ddagger}, Wolfgang Liebetrau{ddagger}, Michael Kazinski{ddagger}, Angelika Bonin-Debs{ddagger}, Christian Behl§, Peter Buckel{ddagger} and Ulrich Brinkmann{ddagger},

From the {ddagger} Xantos Biomedicine AG, Max-Lebsche Platz 31, D-81377 Munich, Germany; and § Institute for Physiological Chemistry and Pathobiochemistry, Johannes-Gutenberg-University Mainz, Duesbergweg 6, D-55099 Mainz, Germany


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We describe a novel genetic screen that is performed by transfecting every individual clone of an expression clone collection into a separate population of cells in a high-throughput mode. We combined high-throughput functional genomics with experimental validation to discover human genes that ameliorate cytotoxic responses of neuronal HT-22 cells upon exposure to oxidative stress. A collection of 5,000 human cDNAs in mammalian expression vectors were individually transfected into HT-22 cells, which were then exposed to H2O2. Five genes were found that are known to be involved in pathways of detoxification of peroxide (catalase, glutathione peroxidase-1, peroxiredoxin-1, peroxiredoxin-5, and nuclear factor erythroid-derived 2-like 2). The presence of those genes in our "hit list" validates our screening platform. In addition, a set of candidate genes was found that has not been previously described as involved in detoxification of peroxide. One of these genes, which was consistently found to reduce H2O2 -induced toxicity in HT-22, was GFPT2. This gene is expressed at significant levels in the central nervous system (CNS) and encodes glutamine-fructose-6-phosphate transaminase (GFPT) 2, a rate-limiting enzyme in hexosamine biosynthesis. GFPT has recently also been shown to ameliorate the toxicity of methylmercury in Saccharomyces cerevisiae. Methylmercury causes neuronal cell death in part by protein modification as well as enhancing the production of reactive oxygen species (ROS). The protective effect of GFPT2 against H2O2 toxicity in neuronal HT-22 cells may be similar to its protection against methylmercury in yeast. Thus, GFPT appears to be conserved among yeast and men as a critical target of methylmercury and ROS-induced cytotoxicity.


The mapping and sequence determination of most human genes, combined with the availability of extensive information regarding genetic factors and/or disease-associated aberrations or variations, has accelerated the search and identification of potential disease-associated genes in the human genome (1, 2). In addition to individual experimental genetic and functional analyses, high-throughput analyses are applied to find disease-associated genes. These "parallel" techniques simultaneously cover many different genes. For example, most human genes can be simultaneously screened for potential disease associations by high-throughput expression profiling (e.g. by mRNA or single nucleotide polymorphism chips), as well as high-throughput in silico analyses of databases that contain genetic as well as clinical/functional information (38). Such databases contain sequences and frequencies of EST sequences and serial analysis of gene expression data from different tissues and disease types, including laser-captured and tissue-specific EST libraries. Mining of databases by bioinformatics techniques (e.g. EST clustering and filtering tools)(7) permits the extraction of novel disease-associated genes (e.g. genes that are found to be significantly lower or higher expressed in diseased versus normal healthy tissues) and hence may cause—or protect against—diseases. Despite the progress made by such bioinformatics procedures, there is a large gap between the amount of sequence and expression information on one hand and the limited experimental information relating to most genes. This discrepancy severely hampers the efficient development of novel targets for the treatment of diseases. The information deficit on functional phenotypes is obvious for many potential target genes relevant for the function and diseases of the central nervous system (CNS).1 Vast data sets related to gene expression profiles, spatially and temporary expression profiles, as well as potential disease associations are available for different regions and/or diseases of the brain. However, experimental validation of the in silico-defined candidate genes is essential. Unfortunately, so far the low-throughput functional analysis is a bottleneck in the process of target and disease gene identification.

To circumvent this bottleneck, we decided to "invert" the currently used process of high-throughput target identification by initiating the screen for novel disease-relevant genes with a high-throughput functional genomics screen as opposed to high-throughput in silico screen (9, 10). The functional screen is aimed at identification of genes by phenotypes that are caused by recombinant expression of the corresponding single cDNAs. For this, we have developed a robotics platform, which performs high-throughput transfection of single cDNAs into mammalian cells, followed by determination of cellular phenotypes (11).

Because of their limited regeneration capacity, damage to nerve cells causes severe disabilities as seen, for example, in Alzheimer’s disease or stroke-reperfusion. Nerve cells are very sensitive toward toxic stimuli (10). Because of that, various mechanisms protect these cells (and the CNS) against poisoning, e.g. the blood brain barrier excludes many poisons from the CNS and diverse detoxification mechanisms exist to counteract damage caused by, for example, reactive oxygen species (ROS). Indeed, ROS-induced oxidative stress has been linked to the processing and progression of a variety of neurodegenerative disorders (12). ROS, generated, for example, by process of Fenton-like reactions (13) in neuronal disorders like Alzheimer’s disease, are efficiently removed by enzymes such as superoxide dismutase, catalase, or glutathione peroxidase. However, up to today, not all pathways and mechanisms that can protect against oxidative damage are known (14). Identification of additional pathways and protective genes may aid in the treatment or prevention of neuronal disease. To identify such novel genes, we have adapted our expression and phenotype determination platform to mimic the deleterious effect of oxidative stress on neuronal cells. Thereby, we identify human genes that can counteract such ROS-induced toxicity. The screen based upon this functional genomics principle resulted in a "hit list" of protective genes that included many known detoxification enzymes and detoxification pathway factors. These included glutathione peroxidase, catalase, perioxiredoxin 1, perioxiredoxin 5, and nuclear factor erythroid-derived 2-like 2. In addition, novel genes were found as hits that have not previously been described as radical detoxification-associated genes.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
High-throughput Functional Screen—
For automated DNA preparation, aliquots containing single cDNA collection clones (OriGene Technologies, Rockville, MD and mammalian gene collection, RZPD, Berlin, Germany) containing human full-length cDNAs in expression vectors (pCMVSport6 (Invitrogen, Carlsbad, CA) or pCMV-XL4 (OriGene)) were grown in 96-well blocks in 1.5 ml of Luria-Bertani/100 µM ampicillin. After incubation at 37 °C for 24 h, bacteria were pelleted by centrifugation, resuspended in 170 µl (50 mM Tris pH 8.0; 10 mM EDTA pH 8.0; 100 µg/ml RNaseA), shaken for 5 min at 1,000 rpm, lysed in 170 µl of 200 mM NaOH; 1% SDS and neutralized by 170 µl of 3 M KAc pH 5.5. Five minutes later, the samples were centrifuged at 3,500 rpm, and the supernatant was transferred and supplied with 120 µl 1.2% SDS in isopropanol and incubated for 20 min at 4 °C. Following centrifugation at 3,500 x g for 10 min, supernatants were transferred to new plates; 120 µl of silica oxide suspension (50 mg/ml) was added and incubated for 5 min at room temperature for DNA binding. The plates were centrifuged at 2000 x g for 5 min, the supernatant was carefully decanted, and 400 µl of acetone was added, mixed for 1 min at 1,000 rpm, and centrifuged at 2,000 x g for 5 min. The pellets were dried at 70 °C and resuspended in 140 µl of water at 65 °C, mixed, and recentrifuged for 5 min at 3,000 x g. Supernatant with plasmid DNA was stored in a 96-well plate at –20 °C. Transfection of HT-22 cells was performed on a transfection robot: ~230 ng of DNA in 10.5 µl was added to single wells in 96-well plates. In another plate, 0.93 µl/well of Metafectene (Biontex, Munich, Germany) was mixed with 35 µl/well Dulbecco’s modified Eagle’s medium (DMEM) w. pyruvate/glutamine. The automated procedure then included addition of 24.5 µl/well of DMEM to the DNA solution to a final volume of 35 µl per well. Thirty-five µl Metafectene/DMEM was added and mixed. After 40 min of incubation at room temperature, 60 µl of Metafectene/DNA solution was added and incubated for 41 h at 37 °C in 5% CO2. Positive clones from both rounds of screening were verified by manual transfection using independent de novo DNA preparations.

ROS Toxicity Studies in HT-22 Cells—
Mouse hippocampal HT-22 (from P. Maher, TSRI, San Diego, CA) were grown in DMEM with penicillin/streptomycin and 1% L-glutamine/pyruvate. HT-22 were seeded on 96-well plates coated with 0.01% poly-L-ornithine for 20 min at a density of 2.0 x 103 cells per well in 100 µl of complete medium using a Multidrop 384 (Thermo Labsystems, Waltham, MA). Twenty-four hours later, the cells were used for transfection. Forty-one hours after transfection, the volume of medium in each well was reduced to a final volume of 50 µl, and 50 µl of double-concentrated t-butyl H2O2 and amphotericin B was added to each well. Eighteen hours later, cells were analyzed by phase-contrast microscopy Axiovert 25 (Zeiss, Oberkochen, Germany). Cell viability was determined with the alamarBlueTM assay (BIOSOURCE, Camarillo, CA). For that, medium volume was reduced to 60 µl, and 50 µl of complete medium supplemented with 10% of alamarBlue reagent was added. Cells were incubated for 3 h at 37 °C, and fluorescence was measured at 590 nm using a Fluoroskan Ascent FL (Thermo Labsystems).


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Screening Assay for Modulators of Oxidative Stress-induced Toxicity—
The goal of this study was to identify novel genes that ameliorate oxidative stress-induced toxicity via application of a high-throughput functional screening method. For that, we developed an assay to detect modulation of H2O2 toxicity in HT-22 cells (Fig. 1). H2O2 toxicity is mediated through an increase of intracellular ROS and a recognized model for oxidative stress-induced toxicity in neuronal cells (13). H2O2 diffuses through membranes and generates highly reactive hydroxyl radicals during Fenton reactions (13). Because HT-22 is an immortalized mouse hippocampal cell line and an established model in oxidative stress (15), we consider the detection of H2O2 toxicity in HT-22 a suitable model for neuronal ROS-dependent toxicity. Fig. 2A shows an example of H2O2 toxicity on HT-22. The cells die in a concentration-dependent manner upon incubation with H2O2.



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FIG. 1. Screening model. Outline of the high-throughput screen for protective cDNAs. Single bacteria clones containing human cDNAs were inoculated in 96-deep-well blocks and grown for 24 h in selection medium. Plasmid DNA was isolated using a protocol with silica oxide (see "Experimental Procedures"). Exponentially dividing HT-22 were plated into 96-well plates. Then every well was transfected with one individual cDNA out of a cDNA collection. After a defined timeline, cells were stimulated with one concentration of H2O2, and survival was assessed using the alamarBlue viability assay.

 


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FIG. 2. Development of assays for identification of genes that ameliorate ROS induced toxicity. A, Dose dependence of H2O2-induced cell death in HT-22 cells after 18 h of incubation. Cells were transfected with glutathione peroxidase-1 or with an empty expression vector (control). Forty hours after transfection, cells were challenged with different concentrations of H2O2; survival was measured 18 h later with an alamarBlue assay. H2O2 did not interact with the alamarBlue assay. The EC50 was 85 µM H2O2. Data were shown as relative fluorescence units, with a mean ± S.D. of three independent measurements (p < 0.001, two-way analysis of variance). B, time course of protection by glutathione peroxidase-1. Cells were transfected with glutathione peroxidase-1 cDNA (left) or with control vector (right). Twenty-four, 31, 41, and 47 h after transfection, cells were challenged with 100 µM H2O2, and cell survival was measured 18 h later via alamarBlue assay. Data are shown as mean ± S.D. of three independent experiments (* p < 0.01 and ** p < 0.001; Student’s t test).

 
To establish a high-throughput assay for the identification of modulators of H2O2-induced toxicity, we needed to combine the H2O2 toxicity assay with transfection and expression of cDNA expression plasmids. To determine a suitable time point of transfection and duration of gene expression in our experimental setup, a known protector for ROS-induced neurotoxicity, glutathione peroxidase-1 (1618), was transfected, and its protective effects in our H2O2 assay were determined at different time points (Fig. 2B). Maximum protective effect by glutathione peroxidase was found when cells were allowed to express the gene product for 41 h followed by the challenge with 90 µM H2O2 for 18 h. Treating cells earlier (24 h, 31 h) with H2O2 strongly reduced the difference in viability between protected and nonprotected cells. This is caused by insufficient amounts of protective gene product being produced in shorter time periods. Longer expression periods lead to reduced sensitivity of the assay using peroxidase as control. Because of that, we chose 41-h expression after transfection followed by the challenge with 90 µM H2O2 for 18 h as general setup for our screening. Under these conditions, we expect to identify protective genes with similar activities and protective "kinetics" such as peroxidase, as well as genes with different modes of action that display protective activities within this assay/time window.

High-throughput Functional Genomics Screen Identified Genes that Ameliorate Oxidative Stress-induced Toxicity—
The dose-dependent cytotoxicity of HT-22 after stimulation with H2O2 combined with expression of cDNAs allowed us to develop a high-throughput functional screen to detect protective cDNAs. Elements of an automated platform composed of robots for DNA preparation from Escherichia coli, transfection and automated phenotype readout were applied for our assay (see "Experimental Procedures"). With this system, it was possible to perform automated experiments in which each well was transfected with one individual cDNA out of a cDNA collection. In a first screening round, we analyzed 5,072 different expression clones originating from two cDNA collections ("Experimental Procedures") in two independent sets of experiments. Double-positive clones were further analyzed. To normalize plate-specific fluctuations of the viability signal of H2O2-challenged cells, we loaded four control wells of each plate with glutathione peroxidase-1 cDNA and vector control, respectively. These controls were also used to prove assay reproducibility. Clones that produced a signal above standard deviation compared with the overall plate mean were defined as hits. This criterion was used for all plate positions except for the rim wells, because the signal deviations in rim wells were generally large (due to evaporation effects). Because of that, hits in rim wells were called if signals were increased to more than the 2-fold value of the overall plate mean. Because this very sensitive "calling" of hits may also generate false positives, the effects of all screening hits were confirmed in independent follow-up experiments. Out of 5,072 clones tested, we identified 97 clones in the first round of screening. Repeating this experiment with all clones in a second round of screening revealed a total of 33 screening hits that showed positivity in both experiments. Thus, the "double-hit" rate in both rounds of screening was 0.7%. The manual retesting of all 33 screening hits unambiguously confirmed the protective effect of six clones (Table I). For these, increased cell viability was consistently observed at two different concentrations of H2O2 in at least three independent experiments. Of the remaining 27 hit candidates that were "double-positive" in the original screen, 10 hits also showed protection against H2O2 in manual reanalyses, albeit with lower protective activities and (therefore) reduced reproducibilities (protection not seen in all experimental repetitions). We therefore excluded these hit candidates from further analyses. It has to be noted, however, that two of these genes have not been assigned to a defined biological function and therefore may encode novel ROS-protective proteins. The identity of these additional weak hits is shown in Table II.


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TABLE I Identified and confirmed human cDNA "hits" that cause ROS protection in HT-2 cells

 

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TABLE II Additional human cDNA hits that were "double-positive" in the ROS protection screen in HT-22 cells and showed activity in manual retesting, however weak and/or not consistently in all experiments

 
Characterization of Hit Genes with a Known Function in ROS Detoxification—
Every round of screening was performed with a defined concentration of H2O2. Because the protective effect of expressed genes in our assay has been shown to depend on the concentration of H2O2 (Fig. 2A), we analyzed the protective effect of all identified hits at different concentrations of H2O2. The dose-response curves and the increased viability mediated by the effects of the six confirmed hits are shown in Fig. 3 (AF). All hits showed a significant shift of EC50 concentration. These experiments demonstrated that our screening approach identified weak (A, B, E, F) as well as strong protectors (C, D). Weak protectors with a known direct function in ROS detoxification were peroxiredoxin-5 and catalase (A, B). The strongest protectors in our setup were peroxiredoxin-1 and glutathione peroxidase-1 (C, D). All of them are known antioxidant enzymes (Table I). The identification of these hits validates the functionality of our screening system for identification of protectors against ROS-mediated cell damage.



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FIG. 3. Protective function of six unambiguously confirmed hits. The cDNAs were identified as hits in both rounds of screening. Exponentially dividing HT-22 cells were plated into 96-well plates. Twenty-four hours later, HT-22 cells were transfected with 230 ng of control or hit plasmid DNA. Forty-one hours later, cells were stimulated with H2O2 for 18 h. Cell viability was measured using the alamarBlue assay. Data are shown as mean ± S.D. of four independent measurements. (A, p < 0.005; and B–F, p < 0.001, two-way analysis of variance).

 
Characterization of Hit Genes with an Indirect or Novel/Unknown Function in Stress Modulation—
Beside antioxidative enzymes, we also identified and confirmed the transcription factor NRF2 (nuclear factor, erythroid-derived 2, like 2; nfe2l2) as protector (Fig. 3E). NRF2 is an antioxidant-responsive element-mediated positive regulator of phase II detoxifying enzymes. It regulates genes that can protect cells against electrophile toxicity, oxidative stress, and carcinogenicity (19). One of the major detoxifying enzymes regulated by NRF2 is glutathione S-transferase, which catalyzes an essential step in glutathione synthesis (20). Another gene that we identified and confirmed as ROS-protective had not previously been described as being involved in detoxification of peroxide-mediated cell damage. This gene, GFPT2, encodes the glutamine-fructose-6-phosphate transaminase (GFPT) 2. GFPT2 is a rate-limiting enzyme in the hexosamine biosynthesis pathway and is mainly expressed in the CNS (21, 22). Our experiments show that transient overexpression of GFPT2 increases viability of HT-22 cells at otherwise toxic concentrations of H2O2 (Fig. 3F).


    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have shown that a combination of high-throughput functional genomics and experimental validation can be applied to discover human genes that ameliorate cytotoxic responses of neuronal HT-22 cells upon exposure to oxidative stress. The presence in our screening hit list of genes that have previously been described to be involved in pathways of detoxification of peroxide (catalase, glutathione peroxidase-1, peroxiredoxin-1, peroxiredoxin-5, and nuclear factor erythroid-derived 2-like 2) validates our screening platform. Additional hits from this screen that were not previously described to be involved in detoxification of peroxide are candidate genes that may be involved in existing and novel pathways of protection against and/or detoxification of ROS. One of these genes that was further analyzed and that was consistently found to reduce H2O2-induced toxicity in HT-22 was GFPT2. This gene encodes GFPT2, a rate-limiting enzyme in hexosamine biosynthesis.

The exact molecular mechanism by which GFPT2 leads to the observed protection against ROS-induced toxicity in HT-22 cells is under investigation. One possible explanation for this protective effect could be that increased expression of GFPT2 activates NF{kappa}B-dependent genes, as shown by James and Scholey (23). Resistance to oxidative stress mediated by NF{kappa}B has been demonstrated (24). Interestingly, GFPT has recently also been shown to ameliorate the toxicity of methylmercury in Saccharomyces cerevisiae (25). The described mode of protection consisted of resupplementation of the rate-limiting enzyme, which becomes inactivated at an essential sulfhydryl group by methylmercury (26). For neuronal cells, it is known that methylmercury causes cell death to cerebellar granule cells and other small neurons in the CNS (27, 28). Here, its mechanism of toxicity is based in part on the depletion of glutathione, protein modifications and raising the intracellular Ca2+ concentration and in enhancing the production of ROS (27, 29). The multiple mode of action of methylmercury (e.g. direct protein modification at sulfhydryl groups as well as an increase of ROS production) and the possibility of overcoming these effects by recombinant supplementation of GFPT in yeast is in full agreement with the observation of GFPT2 being a protector against ROS-induced toxicity in neuronal cells. Thus, GFPT appears to be conserved among yeast and men as a critical target of methylmercury and ROS-induced cytotoxicity.

In general, several applications of the genes isolated by this screen are possible. First, the genes could be used as direct therapeutic agents in gene or protein therapy. Second, this screen complements the information gained from gene chips. Both gene microarray and this screening approach allow us to detect genes that are upregulated. However, as sequences from our screen are already functionally defined, they additionally allow us to establish a causative relation between the gene and the cellular effect. Third, the genes found in this screening approach are putative drug targets that can be used to identify suitable drugs that induce endogenous protective mechanisms.

We envision that combining our high-throughput screening system with various disease-specific cDNA libraries could enhance the yield of new identified gene functions and thereby contribute to the challenge of "post-sequencing" years to link sequences to function.


    ACKNOWLEDGMENTS
 
We thank P. Maher (San Diego) for the HT-22 cells and Katja Duvell, Christine Rottenberger, and Matthias Klein for valuable contributions and Doris Bauer for excellent editorial assistance.


    FOOTNOTES
 
Received, April 22, 2004, and in revised form, June 3, 2004.

Published, MCP Papers in Press, June 4, 2004, DOI 10.1074/mcp.M400054-MCP200

1 The abbreviations used are: CNS, central nervous system; ROS, reactive oxygen species; DMEM, Dulbecco’s modified Eagle’s medium; NRF2, nuclear factor, erythroid-derived 2, like 2; GFPT, glutamine-fructose-6-phosphate transaminase. Back

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Xantos Biomedicine AG, Max-Lebsche-Platz 31, 81377 Munich, Germany. Tel.: 0049-0-89-89959415; Fax: 0049-0-89 89959420; E-mail: u.brinkmann{at}xantos.de


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