The Gpx4NIKO Mouse Is a Versatile Model for Testing Interventions Targeting Ferroptotic Cell Death of Spinal Motor Neurons.

The degeneration and death of motor neurons lead to motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Although the exact mechanism by which motor neuron degeneration occurs is not well understood, emerging evidence implicates the involvement of ferroptosis, an iron-dependent oxidative mode of cell death. We reported previously that treating Gpx4NIKO mice with tamoxifen to ablate the ferroptosis regulator glutathione peroxidase 4 (GPX4) in neurons produces a severe paralytic model resembling an accelerated form of ALS that appears to be caused by ferroptotic cell death of spinal motor neurons. In this study, in support of the role of ferroptosis in this model, we found that the paralytic symptoms and spinal motor neuron death of Gpx4NIKO mice were attenuated by a chemical inhibitor of ferroptosis. In addition, we observed that the paralytic symptoms of Gpx4NIKO mice were malleable and could be tapered by lowering the dose of tamoxifen, allowing for the generation of a mild paralytic model without a rapid onset of death. We further used both models to evaluate mitochondrial reactive oxygen species (mtROS) in the ferroptosis of spinal motor neurons and showed that overexpression of peroxiredoxin 3, a mitochondrial antioxidant defense enzyme, ameliorated symptoms of the mild but not the severe model of the Gpx4NIKO mice. Our results thus indicate that the Gpx4NIKO mouse is a versatile model for testing interventions that target ferroptotic death of spinal motor neurons in vivo.

Enhanced defense against ferroptosis ameliorates cognitive impairment and reduces neurodegeneration in 5xFAD mice.

Oxidative damage including lipid peroxidation is widely reported in Alzheimer's disease (AD) with the peroxidation of phospholipids in membranes being the driver of ferroptosis, an iron-dependent oxidative form of cell death. However, the importance of ferroptosis in AD remains unclear. This study tested whether ferroptosis inhibition ameliorates AD. 5xFAD mice, a widely used AD mouse model with cognitive impairment and robust neurodegeneration, exhibit markers of ferroptosis including increased lipid peroxidation, elevated lyso-phospholipids, and reduced level of Gpx4, the master defender against ferroptosis. To determine if enhanced defense against ferroptosis retards disease development, we generated 5xFAD mice that overexpress Gpx4, i.e., 5xFAD/GPX4 mice. Consistent with enhanced defense against ferroptosis, neurons from 5xFAD/GPX4 mice showed an augmented capacity to reduce lipid reactive oxygen species. In addition, compared with control 5xFAD mice, 5xFAD/GPX4 mice showed significantly improved learning and memory abilities and had reduced neurodegeneration. Moreover, 5xFAD/GPX4 mice exhibited attenuated markers of ferroptosis. Our results indicate that enhanced defense against ferroptosis is effective in ameliorating cognitive impairment and decreasing neurodegeneration of 5xFAD mice. The findings support the notion that ferroptosis is a key contributor to AD pathogenesis.

Overexpression of ferroptosis defense enzyme Gpx4 retards motor neuron disease of SOD1G93A mice.

Degeneration and death of motor neurons in Amyotrophic Lateral Sclerosis (ALS) are associated with increased lipid peroxidation. Lipid peroxidation is the driver of ferroptosis, an iron-dependent oxidative mode of cell death. However, the importance of ferroptosis in motor neuron degeneration of ALS remains unclear. Glutathione peroxidase 4 (Gpx4) is a key enzyme in suppressing ferroptosis by reducing phospholipid hydroperoxides in membranes. To assess the effect of increased protection against ferroptosis on motor neuron disease, we generated SOD1G93AGPX4 double transgenic mice by cross-breeding GPX4 transgenic mice with SOD1G93A mice, a widely used ALS mouse model. Compared with control SOD1G93A mice, both male and female SOD1G93AGPX4 mice had extended lifespans. SOD1G93AGPX4 mice also showed delayed disease onset and increased motor function, which were correlated with ameliorated spinal motor neuron degeneration and reduced lipid peroxidation. Moreover, cell toxicity induced by SOD1G93A was ameliorated by Gpx4 overexpression and by chemical inhibitors of ferroptosis in vitro. We further found that the anti-ferroptosis defense system in spinal cord tissues of symptomatic SOD1G93A mice and sporadic ALS patients might be compromised due to deficiency of Gpx4. Thus, our results suggest that ferroptosis plays a key role in motor neuron degeneration of ALS.

Ablation of ferroptosis regulator glutathione peroxidase 4 in forebrain neurons promotes cognitive impairment and neurodegeneration.

Synaptic loss and neuron death are the underlying cause of neurodegenerative diseases such as Alzheimer's disease (AD); however, the modalities of cell death in those diseases remain unclear. Ferroptosis, a newly identified oxidative cell death mechanism triggered by massive lipid peroxidation, is implicated in the degeneration of neurons populations such as spinal motor neurons and midbrain neurons. Here, we investigated whether neurons in forebrain regions (cerebral cortex and hippocampus) that are severely afflicted in AD patients might be vulnerable to ferroptosis. To this end, we generated Gpx4BIKO mouse, a mouse model with conditional deletion in forebrain neurons of glutathione peroxidase 4 (Gpx4), a key regulator of ferroptosis, and showed that treatment with tamoxifen led to deletion of Gpx4 primarily in forebrain neurons of adult Gpx4BIKO mice. Starting at 12 weeks after tamoxifen treatment, Gpx4BIKO mice exhibited significant deficits in spatial learning and memory function versus Control mice as determined by the Morris water maze task. Further examinations revealed that the cognitively impaired Gpx4BIKO mice exhibited hippocampal neurodegeneration. Notably, markers associated with ferroptosis, such as elevated lipid peroxidation, ERK activation and augmented neuroinflammation, were observed in Gpx4BIKO mice. We also showed that Gpx4BIKO mice fed a diet deficient in vitamin E, a lipid soluble antioxidant with anti-ferroptosis activity, had an expedited rate of hippocampal neurodegeneration and behavior dysfunction, and that treatment with a small-molecule ferroptosis inhibitor ameliorated neurodegeneration in those mice. Taken together, our results indicate that forebrain neurons are susceptible to ferroptosis, suggesting that ferroptosis may be an important neurodegenerative mechanism in diseases such as AD.

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

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.

NLRP3 inflammasome activation by mitochondrial reactive oxygen species plays a key role in long-term cognitive impairment induced by paraquat exposure.

Exposure to environmental toxins such as pesticides is implicated in increasing Alzheimer's disease risk. In this study, we investigated the long-term effects of paraquat exposure on cognition of Alzheimer's disease animal model APP/PS1 mice and wild-type (WT) mice. Our results showed that APP/PS1 mice had exacerbated cognition impairment and elevated Aß levels at 5 months after paraquat exposure, and that WT mice had cognition impairment at 5 and 16 months after paraquat exposure. In addition, increased mitochondrial oxidative stress and augmented brain inflammation were observed in both paraquat-exposed APP/PS1 mice and WT mice. Interestingly, activation of NLRP3 inflammasome, which triggers inflammation in response to mitochondrial stress, was enhanced in paraquat-exposed mice. Moreover, transgenic mice overexpressing Prdx3, a key enzyme in detoxifying mitochondrial H2O2, had suppressed NLRP3 inflammasome activation, reduced brain inflammation, and attenuated cognition impairment after paraquat exposure. Together, our results indicate that NLRP3 inflammasome activation induced by mitochondrial reactive oxygen species plays a key role in mediating paraquat-induced long-term cognition decline by elevating brain inflammation.

Enhanced defense against mitochondrial hydrogen peroxide attenuates age-associated cognition decline.

Increased mitochondrial hydrogen peroxide (H2O2) is associated with Alzheimer's disease and brain aging. Peroxiredoxin 3 (Prdx3) is the key mitochondrial antioxidant defense enzyme in detoxifying H2O2. To investigate the importance of mitochondrial H2O2 in age-associated cognitive decline, we compared cognition between aged (17-19 months) APP transgenic mice and APP/Prdx3 double transgenic mice (dTG) and between old (24 months) wild-type mice and Prdx3 transgenic mice (TG). Compared with aged APP mice, aged dTG mice showed improved cognition that was correlated with reduced brain amyloid beta levels and decreased amyloid beta production. Old TG mice also showed significantly increased cognitive ability compared with old wild-type mice. Both aged dTG mice and old TG mice had reduced mitochondrial oxidative stress and increased mitochondrial function. Moreover, CREB signaling, a signaling pathway important for cognition was enhanced in both aged dTG mice and old TG mice. Thus, our results indicate that mitochondrial H2O2 is a key culprit of age-associated cognitive impairment, and that a reduction of mitochondrial H2O2 could improve cognition by maintaining mitochondrial health and enhancing CREB signaling.

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.

Cognitive impairment and increased Aß levels induced by paraquat exposure are attenuated by enhanced removal of mitochondrial H(2)O(2).

Pesticide exposure is a risk factor of Alzheimer's disease (AD). However, little is known about how pesticide exposure may promote AD pathogenesis. In this study, we investigated the effects of paraquat pesticide exposure on ß-amyloid (Aß) levels and cognition using wild-type (WT) mice and ß-amyloid precursor protein (APP) transgenic mice. Our results showed that wild-type mice and APP transgenic mice after paraquat exposure had increased oxidative damage specifically in mitochondria of cerebral cortex and exhibited mitochondrial dysfunction. Moreover, the elevated mitochondrial damage was directly correlated with impaired associative learning and memory and increased Aß levels in APP transgenic mice exposed to paraquat. Furthermore, overexpression of peroxiredoxin 3, a mitochondrial antioxidant defense enzyme important for H(2)O(2) removal, protected against paraquat-induced mitochondrial damage and concomitantly improved cognition and decreased Aß levels in APP transgenic mice. Therefore, our results demonstrate that mitochondrial damage is a key mechanism underlying cognitive impairment and elevated amyloidogenesis induced by paraquat and that enhanced removal of mitochondrial H(2)O(2) could be an effective strategy to ameliorate AD pathogenesis induced by pesticide exposure.

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

H(2)O(2) 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 H(2)O(2) remain poorly understood. Peroxiredoxin 3 (Prdx3/Prx3) is a thioredoxin peroxidase localized in mitochondria. To understand the cellular and physiological roles of mitochondrial H(2)O(2) 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 H(2)O(2), 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 H(2)O(2) by overexpressing Prdx3 improves glucose tolerance.

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 beta-secretase activity than their age-matched wildtype controls, and the increased beta-secretase activity in Gpx4+/- mice was a result of up-regulation of beta-site amyloid precursor protein cleavage enzyme 1 (BACE1) expression at the protein level. The high level of BACE1 protein led to increased endogenous beta-amyloid (Abeta)(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 Abeta(1-40) and Abeta(1-42) 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.

Accumulation of oxidatively generated DNA damage in the brain: a mechanism of neurotoxicity.

Unrepaired or erroneously repaired DNA lesions drive genomic instability and contribute to cellular and organ decline. Since delayed neuropathologies are common in survivors of smoke inhalation injuries, we asked whether the integrity of brain DNA might be compromised by acute exposure to combustion smoke. Although many studies demonstrate that the brain is equipped to repair oxidatively damaged DNA, to date, the capacity for accurate DNA repair under conditions of disrupted oxygenation and oxidative stress has not been defined. We show that DNA adducts detectable by their ability to block PCR amplification form in the rat hippocampus after acute exposure to smoke. To identify the different types of adducts and to dissect their temporal formation and repair profiles in vivo in the brain, we used DNA-modifying enzymes to convert specific adducts into strand breaks prior to PCR amplification. Using this strategy, we detected formation of oxidative DNA adducts early on after smoke inhalation, while mismatched bases emerged at the later recovery times, potentially due to an erroneous DNA repair process. Erroneous repair can be mutagenic and because the initial smoke-induced oxidative damage to DNA is extensive, compromised fidelity of DNA repair may underlie neurotoxicity and contribute to delayed death of hippocampal neurons.

Melatonin protects against MPTP/MPP+ -induced mitochondrial DNA oxidative damage in vivo and in vitro.

The effects of melatonin on the mitochondrial DNA (mtDNA) damage induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine ion (MPP(+)) were investigated both in vivo and in vitro. MPTP (24 mg/kg, s.c.) induced a rapid increase in the immunoreactivity of 8-hydroxyguanine (8-oxoG), a common biomarker of DNA oxidative damage, in the cytoplasm of neurons in the Substantia Nigra Compact of mouse brain. Melatonin preinjection (7.5, 15 or 30 mg/kg, i.p.) dose-dependently prevented MPTP-induced DNA oxidative damage. In SH-SY5Y cells, MPP(+) (1 mm) increased the immunoreactivity of 8-oxoG in the mitochondria at 1 hr and in the nucleus at 3 hr after treatment. Melatonin (200 microm) preincubation significantly attenuated MPP(+)-induced mtDNA oxidative damage. Furthermore, MPP(+) time-dependently increased the accumulation of mitochondrial oxygen free radicals (mtOFR) from 1 to 24 hr and gradually decreased the mitochondrial membrane potential (Psim) from 18 to 36 hr after incubation. At 72 hr after incubation, MPP(+) caused cell death in 49% of the control. However, melatonin prevented MPP(+)-induced mtOFR generation and Psim collapse, and later cell death. The present results suggest that cytoprotection of melatonin against MPTP/MPP(+)-induced cell death may be associated with the attenuation of mtDNA oxidative damage via inhibition of mtOFR generation and the prevention of Psim collapse.

Effect of tea catechins on the change of glutathione levels caused by Pb(++) in PC12 cells.

Our previous research showed that tea catechins could significantly increase the viability of lead-exposed PC12 cells. Whereas the cellular thiol status is known to be responsible for protecting against lead-induced toxicity, whether the role of tea catechins on lead-induced PC12 cell toxicity is related to the metabolism of intracellular thiol compounds remained vague. In the present study, it was found that Pb(2+) significantly decreased reduced glutathione (GSH)/oxidative glutathione (GSSG) and protein sulfhydryl groups (PSH)/glutathione-protein mixed disulfide (GSSP) ratios as well as glutathione reductase activities in a concentration-dependent manner. Both (-)-epicatechin and (-)-epicatechin gallate (ECG) supplementation resulted in an increased GSH/GSSG ratio and glutathione reductase activities. The galloylated catechins (ECG or (-)-epigallocatechin gallate) treatment significantly decreased the GSSP levels and increased the intracellular PSH/GSSP ratio in lead-exposed PC12 cells. To our surprise, as compared with the group treated by lead acetate, 100 microM EGC treatment following lead exposure significantly decreased GSH/GSSG and PSH/GSSP ratios, as well as glutathione reductase activities. The results suggested that the effect of tea catechins on the intracellular thiols status in PC12 cells was different, which may be related to their chemical structures and/or regulation of special gene expression properties.

Tea catechins protect against lead-induced ROS formation, mitochondrial dysfunction, and calcium dysregulation in PC12 cells.

Recent studies have shown that lead causes oxidative stress by inducing the generation of reactive oxygen species (ROS) and reducing the antioxidant defense system of cells, which suggests that antioxidants may play an important role in the treatment of lead poisoning. The present study was designed to elucidate whether tea catechins had any protective effects on altered oxidative stress parameter in PC12 cells exposed to lead. The experimental results showed that lead decreased PC12 cell viability and induced a rapid elevation of [Ca(2+)](i), which was followed by an accumulation of ROS and a decrease of mitochondrial membrane potential (MMP). Treatment by tea catechins significantly increased cell viability, decreased intracellular Ca(2+) levels and ROS formation, and improved MMP in PC12 cells exposed to lead. The galloylated catechins showed a greater effect on ROS formation and mitochondrial dysfunction than that of nongalloylated catechins, which was similar to the result of their scavenging ability on free radical. In view of the time course of ROS formation and mitochondrial dysfunction and their correlation, our results also suggested that the beneficial effects of tea catechins on MMP are related, at least in part, to its ability to scavenge ROS in PC12 cells exposed to 100 microM Pb(2+). The present results suggest that tea catechins supplementation may play a role for modulating oxidative stress in PC12 cells exposed to lead.

Tea catechins protect against lead-induced cytotoxicity, lipid peroxidation, and membrane fluidity in HepG2 cells.

Recent studies have shown that lead causes oxidative stress by inducing the generation of reactive oxygen species (ROS) and reducing the antioxidant defense system of cells. This suggests that antioxidants may play an important role in the treatment of lead poisoning as a kind of excellent scavenger of free radicals and chelator of heavy metal. Whether tea catechins have protective effects against oxidative stress after lead treatment in cell systems remains unclear. The present study was designed to elucidate if tea catechins have any protective effects on lipid peroxidation damage in lead-exposed HepG2 cells. Exposure of HepG2 cells to Pb(++) decreased cell viability and stimulated lipid peroxidation of cell membranes as measured by the thioburbituric acid reaction. Electron spin resonance (ESR) spin-labeling studies indicated that lead exposure could decrease the fluidity in the polar surface of cell membranes. Tea catechin treatment significantly increased cell viability, decreased lipid peroxidation levels, and protected cell membrane fluidity in lead-exposed HepG2 cells in a concentration-dependent manner. The galloylated catechins showed a stronger effect than nongalloylated catechins. Cotreatment with (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) showed a synergistically protective effect. The results suggest that tea catechin supplementation may have a role to play in modulating oxidative stress in lead-exposed HepG2 cells.