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

Abstract

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 highthroughput 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 H 2 O 2 . 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 H 2 O 2 -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 H 2 O 2 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.

Changes in granulosa cells' gene expression associated with increased oocyte competence in bovine

“…GFPT2 is known to reduce peroxide toxicity associated with oxidative stress (Zitzler et al 2004). TF is implicated in iron transport that is positively related to oxidative stress (conversion of peroxide to OH.)…”

Proteomic analysis of oxidative stress-induced neuronal cell death by using two-dimensional fluorescence difference gel electrophoresis

“…Previously, it was reported that peroxiredoxin 1 and 5 have been shown to ameliorate ROS-induced cytotoxicity (18). These results clearly showed that the defense system involving antioxidant enzymes against oxidative stress is activated by glutamate-induced stress.…”

“…HSP70 is known to inhibit apoptosis by neutralizing and interacting with AIF (36). Overexpression of HSP70 also ameliorates glutamate-induced cell death in HT22 cells (18). More extensive studies are needed to elucidate whether HSP70 interacts with AIF or if AIF has certain functional roles in glutamate-induced cell death.…”

High throughput functional genomics: Identification of novel genes with tumor suppressor phenotypes

“…15,16 The pipeline (Fig. 1a) begins with individual cDNA expression plasmids from which are propagated (by Robot 1) in bacteria and isolated by a high throughput Robot 2 (20,000 preps per 24 hr) that delivers reproducible quantities of plasmids without contaminations that interfere with subsequent transfection assays.…”

“…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 such as proliferation and cell death. 15,16 Because the balance between proliferation and apoptosis is crucial for normal development and homeostasis, 17,18 genes or genetic alterations that interfere with this balance cause diseases, e.g., cancer. 8,9 Our expression and phenotype-determination platform can mimic gain of function deregulation and analyze the resulting phenotype.…”