Qitao Ran

Is the Oxidative Stress Theory of Aging Dead

Currently, the oxidative stress (or free radical) theory of aging is the most popular explanation of how aging occurs at the molecular level. While data from studies in invertebrates (e.g., C. elegans and Drosophila) and rodents show a correlation between increased lifespan and resistance to oxidative stress (and in some cases reduced oxidative damage to macromolecules), direct evidence showing that alterations in oxidative damage/stress play a role in aging are limited to a few studies with transgenic Drosophila that overexpress antioxidant enzymes. Over the past eight years, our laboratory has conducted an exhaustive study on the effect of under- or overexpressing a large number and wide variety of genes coding for antioxidant enzymes. In this review, we present the survival data from these studies together. Because only one (the deletion of the Sod1 gene) of the 18 genetic manipulations we studied had an effect on lifespan, our data calls into serious question the hypothesis that alterations in oxidative damage/stress play a role in the longevity of mice.

THE SELENOPROTEIN GPX4 IS ESSENTIAL FOR MOUSE DEVELOPMENT AND PROTECTS FROM RADIATION AND OXIDATIVE DAMAGE INSULTS

Lipid peroxidation has been implicated in a variety of pathophysiological processes, including inflammation, atherogenesis, neurodegeneration, and the ageing process. Phospholipid hydroperoxide glutathione peroxidase (GPX4) is the only major antioxidant enzyme known to directly reduce phospholipid hydroperoxides within membranes and lipoproteins, acting in conjunction with alpha tocopherol (vitamin E) to inhibit lipid peroxidation. Here we describe the generation and characterization of GPX4-deficient mice by targeted disruption of the murine Gpx4 locus through homologous recombination in embryonic stem cells. Gpx4(-/-) embryos die in utero by midgestation (E7.5) and are associated with a lack of normal structural compartmentalization. Gpx4(+/-) mice display reduced levels of Gpx4 mRNA and protein in various tissues. Interestingly, cell lines derived from Gpx4(+/-) mice are markedly sensitive to inducers of oxidative stress, including gamma-irradiation, paraquat, tert-butylhydroperoxide, and hydrogen peroxide, as compared to cell lines derived from wild-type control littermates. Gpx4(+/-) mice also display reduced survival in response to gamma-irradiation. Our observations establish GPX4 as an essential antioxidant enzyme in mice and suggest that it performs broad functions as a component of the mammalian antioxidant network.

Transgenic Mice Overexpressing Glutathione Peroxidase 4 Are Protected against Oxidative Stress-induced Apoptosis

Glutathione peroxidase 4 (Gpx4) is uniquely involved in the detoxification of oxidative damage to membrane lipids. Our previous studies showed that Gpx4 is essential for mouse survival and that Gpx4 deficiency makes cells vulnerable to oxidative injury. In the present study, we generated two lines of transgenic mice overexpressing Gpx4 (Tg(GPX4) mice) using a genomic clone containing the human GPX4 gene. Both lines of Tg-(GPX4) mice, Tg5 and Tg6, had elevated levels of Gpx4 (mRNA and protein) in all tissues investigated, and overexpression of Gpx4 did not cause alterations in activities of glutathione peroxidase 1, catalase, Cu/Zn superoxide dismutase, and manganese superoxide dismutase. The human GPX4 transgene rescued the lethal phenotype of null mutation of the mouse Gpx4 gene, indicating that the transgene can replace the essential role of mouse Gpx4 in mouse development. Cell death induced by t-butylhydroperoxide and diquat was significantly less in murine embryonic fibroblasts from Tg(GPX4) mice compared with wild type mice. Liver damage and lipid peroxidation induced by diquat were reduced significantly in Tg(GPX4) mice. In addition, diquat-induced apoptosis was decreased in Tg(GPX4) mice, as evidenced by attenuated caspase-3 activation and reduced cytochrome c release from mitochondria. These data demonstrate that Gpx4 plays a role in vivo in the mechanism of apoptosis induced by oxidative stress that most likely occurs through oxidative damage to mitochondrial phospholipids such as cardiolipin.

Identification of a Gene That Reverses the Immortal Phenotype of a Subset of Cells and Is a Member of a Novel Family of Transcription Factor-Like Genes

ABSTRACT Based on the dominance of cellular senescence over immortality, immortal human cell lines have been assigned to four complementation groups for indefinite division. Human chromosomes carrying senescence genes have been identified, including chromosome 4. We report the cloning and identification of a gene, mortality factor 4 (MORF 4), which induces a senescent-like phenotype in immortal cell lines assigned to complementation group B with concomitant changes in two markers for senescence. MORF 4 is a member of a novel family of genes with transcription factor-like motifs. We present here the sequences of the seven family members, their chromosomal locations, and a partial characterization of the three members that are expressed. Elucidation of the mechanism of action of these genes should enhance our understanding of growth regulation and cellular aging.

Reduction of mitochondrial H2O2 by overexpressing peroxiredoxin 3 improves glucose tolerance in mice.

H2O2 is a major reactive oxygen species produced by mitochondria that is implicated to be important in aging and pathogenesis of diseases such as diabetes; however, the cellular and physiological roles of mitochondrial H2O2 remain poorly understood. Peroxiredoxin 3 (Prdx3/Prx3) is a thioredoxin peroxidase localized in mitochondria. To understand the cellular and physiological roles of mitochondrial H2O2 in aging and pathogenesis of age‐associated diseases, we generated transgenic mice overexpressing Prdx3 (Tg(PRDX3) mice). Tg(PRDX3) mice overexpress Prdx3 in a broad range of tissues, and the Prdx3 overexpression occurs exclusively in the mitochondria. As a result of increased Prdx3 expression, mitochondria from Tg(PRDX3) mice produce significantly reduced amount of H2O2, and cells from Tg(PRDX3) mice have increased resistance to stress‐induced cell death and apoptosis. Interestingly, Tg(PRDX3) mice show improved glucose homeostasis, as evidenced by their reduced levels of blood glucose and increased glucose clearance. Tg(PRDX3) mice are also protected against hyperglycemia and glucose intolerance induced by high‐fat diet feeding. Our results further show that the inhibition of GSK3 may play a role in mediating the improved glucose tolerance phenotype in Tg(PRDX3) mice. Thus, our results indicate that reduction of mitochondrial H2O2 by overexpressing Prdx3 improves glucose tolerance.

Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease

Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides to lethal levels. Emerging evidence suggests that ferroptosis represents an ancient vulnerability caused by the incorporation of polyunsaturated fatty acids into cellular membranes, and cells have developed complex systems that exploit and defend against this vulnerability in different contexts. The sensitivity to ferroptosis is tightly linked to numerous biological processes, including amino acid, iron, and polyunsaturated fatty acid metabolism, and the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. Ferroptosis has been implicated in the pathological cell death associated with degenerative diseases (i.e., Alzheimers, Huntingtons, and Parkinsons diseases), carcinogenesis, stroke, intracerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and kidney degeneration in mammals and is also implicated in heat stress in plants. Ferroptosis may also have a tumor-suppressor function that could be harnessed for cancer therapy. This Primer reviews the mechanisms underlying ferroptosis, highlights connections to other areas of biology and medicine, and recommends tools and guidelines for studying this emerging form of regulated cell death.

Lipid peroxidation up-regulates BACE1 expression in vivo : a possible early event of amyloidogenesis in Alzheimer's disease

Increased lipid peroxidation is shown to be an early event of Alzheimer’s disease (AD). However, it is not clear whether and how increased lipid peroxidation might lead to amyloidogenesis, a hallmark of AD. Glutathione peroxidase 4 (Gpx4) is an essential antioxidant defense enzyme that protects an organism against lipid peroxidation. Gpx4+/− mice show increased lipid peroxidation in brain, as evidenced by their elevated levels of 4‐hydroxy‐2‐nonenal. To understand the role of lipid peroxidation in amyloidogenesis, we studied secretase activities in Gpx4+/− mice as a function of age. Both young (6 months) and middle‐aged (17–20 months) Gpx4+/− mice had higher levels of β‐secretase activity than their age‐matched wildtype controls, and the increased β‐secretase activity in Gpx4+/− mice was a result of up‐regulation of β‐site amyloid precursor protein cleavage enzyme 1 (BACE1) expression at the protein level. The high level of BACE1 protein led to increased endogenous β‐amyloid (Aβ)1–40 in middle‐aged Gpx4+/− mice. We further studied amyloidogenesis in APPGpx4+/− mice. Our data indicate that APPGpx4+/− mice had significantly increased amyloid plaque burdens and increased Aβ1–40 and Aβ1–42 levels compared with APPGpx4+/+ mice. Therefore, our results indicate that increased lipid peroxidation leads to increased amyloidogenesis through up‐regulation of BACE1 expression in vivo, a mechanism that may be important in pathogenesis of AD at early stages.

Glutathione peroxidase 4 protects cortical neurons from oxidative injury and amyloid toxicity

Polyunsaturated fatty acids (PUFA) in membrane lipids are prone to attack by reactive oxygen species (ROS), and the resulting lipid peroxidation can cause injury and death of cells. Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme that can directly detoxify lipid hydroperoxides generated by ROS. Overexpression of Gpx4 has been shown to be protective against oxidative damage in several cell lines. We examined in this study the stress response of neurons with increased expression of Gpx4, because neurons are especially vulnerable to oxidative injury as a result of their high content of PUFA. Our results show that primary culture cortical neurons derived from Gpx4 transgenic mice, which had increased expression of Gpx4, had increased cell survival and reduced level of apoptosis after exposure to t‐butyl hydroperoxide and hydrogen peroxide. We also studied the protective role of Gpx4 against β‐amyloid toxicity, because β‐amyloid‐induced neural toxicity is believed to be mediated through lipid peroxidation. Primary culture cortical neurons from Gpx4 transgenic mice had significantly less cell toxicity than their wild‐type counterparts after exposure to Aβ25–35 and Aβ1–40 peptides, and apoptosis induced by Aβ25–35 was attenuated in neurons from Gpx4 transgenic mice. Our data demonstrate that overexpression of Gpx4 protects neurons against oxidative injury and β‐amyloid‐induced cytotoxicity.

Ablation of the Ferroptosis Inhibitor Glutathione Peroxidase 4 in Neurons Results in Rapid Motor Neuron Degeneration and Paralysis.

Background: Glutathione peroxidase 4 (GPX4) is shown to be a key inhibitor of ferroptosis, a cell death mechanism involving lipid reactive oxygen species. Results: Conditional ablation of Gpx4 in neurons resulted in rapid motor neuron degeneration and paralysis in mice. Conclusion: Lack of GPX4 triggered motor neuron degeneration characterized by ferroptosis. Significance: Ferroptosis inhibition may be essential for motor neuron health and survival in vivo. Glutathione peroxidase 4 (GPX4), an antioxidant defense enzyme active in repairing oxidative damage to lipids, is a key inhibitor of ferroptosis, a non-apoptotic form of cell death involving lipid reactive oxygen species. Here we show that GPX4 is essential for motor neuron health and survival in vivo. Conditional ablation of Gpx4 in neurons of adult mice resulted in rapid onset and progression of paralysis and death. Pathological inspection revealed that the paralyzed mice had a dramatic degeneration of motor neurons in the spinal cord but had no overt neuron degeneration in the cerebral cortex. Consistent with the role of GPX4 as a ferroptosis inhibitor, spinal motor neuron degeneration induced by Gpx4 ablation exhibited features of ferroptosis, including no caspase-3 activation, no TUNEL staining, activation of ERKs, and elevated spinal inflammation. Supplementation with vitamin E, another inhibitor of ferroptosis, delayed the onset of paralysis and death induced by Gpx4 ablation. Also, lipid peroxidation and mitochondrial dysfunction appeared to be involved in ferroptosis of motor neurons induced by Gpx4 ablation. Taken together, the dramatic motor neuron degeneration and paralysis induced by Gpx4 ablation suggest that ferroptosis inhibition by GPX4 is essential for motor neuron health and survival in vivo.

Gpx4 ablation in adult mice results in a lethal phenotype accompanied by neuronal loss in brain

Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme important in reducing hydroperoxides in membrane lipids and lipoproteins. Gpx4 is essential for survival of embryos and neonatal mice; however, whether Gpx4 is required for adult animals remains unclear. In this study, we generated a floxed Gpx4 mouse (Gpx4(f/f)), in which exons 2-4 of Gpx4 gene are flanked by loxP sites. We then cross-bred the Gpx4(f/f) mice with a tamoxifen (tam)-inducible Cre transgenic mouse (R26CreER mice) to obtain mice in which the Gpx4 gene could be ablated by tam administration (Gpx4(f/f)/Cre mice). After treatment with tam, adult Gpx4(f/f)/Cre mice (6-9 months of age) showed a significant reduction of Gpx4 levels (a 75-85% decrease) in tissues such as brain, liver, lung, and kidney. Tam-treated Gpx4(f/f)/Cre mice lost body weight and died within 2 weeks, indicating that Gpx4 is essential for survival of adult animals. Tam-treated Gpx4(f/f)/Cre mice exhibited increased mitochondrial damage, as evidenced by the elevated 4-hydroxylnonenal (4-HNE) level, decreased activities of electron transport chain complexes I and IV, and reduced ATP production in liver. Tam treatment also significantly elevated apoptosis in Gpx4(f/f)/Cre mice. Moreover, tam-treated Gpx4(f/f)/Cre mice showed neuronal loss in the hippocampus region and had increased astrogliosis. These data indicate that Gpx4 is essential for mitochondria integrity and survival of neurons in adult animals.