Biomedical subjects

Peter J Crack

Publications and source records attributed to Peter J Crack.

14 recordsLinked to original sources

PubMed2006

Glutathione peroxidase 1 and glutathione are required to protect mouse astrocytes from iron-mediated hydrogen peroxide toxicity.

The enzyme glutathione peroxidase 1 (GPx1) is involved in the cellular detoxification of peroxides. To test for the consequences of GPx deficiency in astrocytes, astrocyte-rich primary cultures from wild-type and GPx1-deficient [GPx1(-/-)] mice were exposed to H(2)O(2). In GPx1(-/-) astrocytes, the clearance rate of H(2)O(2) was slower than in wild-type cells. In contrast to GPx1-deficient astrocytes, wild-type cells exhibited, within 2 min of H(2)O(2) application, a rapid and transient accumulation of cellular glutathione disulfide that amounted to 60% of total glutathione. The peroxide treatment did not affect the viability of wild-type astrocytes, whereas 45% of the GPx1(-/-) cells died within 8 hr. However, the viability of both types of astrocytes was strongly compromised by lowering cellular glutathione content before peroxide application. In contrast, inactivation of catalase caused substantial cell death only in GPx1(-/-) cells but not in wild-type astrocytes. The cell death observed was prevented by the iron chelators deferoxamine, 1,10-phenathroline, or 2,2'-dipyridyl, whereas preincubation with ferric ammonium citrate increased the toxicity of peroxide treatments. These results demonstrate that GPx1 contributes to the rapid clearance of H(2)O(2) by mouse astrocytes and that both GPx1 and a high concentration of glutathione are required to protect these cells from iron-dependent peroxide damage.

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PubMed2006

Potential contribution of NF-kappaB in neuronal cell death in the glutathione peroxidase-1 knockout mouse in response to ischemia-reperfusion injury.

BACKGROUND AND PURPOSE: We have previously identified an increased susceptibility of Gpx1(-/-) mice to increased infarct size after middle cerebral artery occlusion (MCAO). This study was designed to elucidate the mechanisms involved in elevated neuronal cell death arising from an altered endogenous oxidant state. METHODS: Gpx1(-/-) mice were exposed to transient MCAO and reperfusion by intraluminal suture blockade. Protein expression of the p65 subunit of transcription factor nuclear factor-kappaB (NF-kappaB) was examined by immunohistochemistry and Western Analysis. NF-kappaB DNA-protein activity was assessed by electrophoretic mobility shift assays (EMSA). Wild-type and Gpx1(-/-) mice were exposed to MCAO with or without the NF-kappaB inhibitor, pyrrolidinedithiocarbamate (PDTC). RESULTS: Upregulation of the p65 subunit of NF-kappaB and subsequent p65 phosphorylation at serine 536 was detected in the Gpx1(-/-) brains after stroke. EMSAs revealed that increased ischemia-enhanced DNA binding of NF-kappaB was observed in Gpx1(-/-) mice compared with wild-type. Supershift assays indicated that the p50 and p65 subunits participated in the bound NF-kappaB complex. The NF-kappaB inhibitor PDTC, a potential antioxidant, was able to afford partial neuroprotection in the Gpx1(-/-) mice. CONCLUSIONS: NF-kappaB is upregulated in the Gpx1(-/-) mouse, and this upregulation contributes to the increased cell death seen in the Gpx1(-/-) after MCAO. The activation of NF-kappaB may increase the expression of downstream target genes that are involved in the progression of neural injury after MCAO.

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PubMed2006

Suppressor of cytokine signaling 1 negatively regulates Toll-like receptor signaling by mediating Mal degradation.

Toll-like receptor (TLR) signals that initiate innate immune responses to pathogens must be tightly regulated to prevent excessive inflammatory damage to the host. The adaptor protein Mal is specifically involved in signaling via TLR2 and TLR4. We demonstrate here that after TLR2 and TLR4 stimulation Mal becomes phosphorylated by Bruton's tyrosine kinase (Btk) and then interacts with SOCS-1, which results in Mal polyubiquitination and subsequent degradation. Removal of SOCS-1 regulation potentiates Mal-dependent p65 phosphorylation and transactivation of NF-kappaB, leading to amplified inflammatory responses. These data identify a target of SOCS-1 that regulates TLR signaling via a mechanism distinct from an autocrine cytokine response. The transient activation of Mal and subsequent SOCS-1-mediated degradation is a rapid and selective means of limiting primary innate immune response.

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PubMed2006

Glutathione peroxidase 1 and a high cellular glutathione concentration are essential for effective organic hydroperoxide detoxification in astrocytes.

Organic hydroperoxides are produced in the eicosanoid metabolism and by lipid peroxidation. To examine the contribution of glutathione peroxidase-1 (GPx1) and glutathione (GSH) in the disposal of organic hydroperoxides in brain astrocytes, primary astrocyte cultures from wild type or GPx1-deficient (GPx1(-/-)) mice were exposed to cumene hydroperoxide (CHP). After application of 100 microM CHP, the peroxide disappeared quickly from the incubation medium of wild type cells with a half-life of 9 min, whereas CHP clearance was strongly retarded in GPx1(-/-) astrocytes. Depletion of GSH by pre-incubation with buthionine sulfoximine (BSO) significantly slowed CHP clearance by wild type astrocytes, while almost completely preventing peroxide disposal by GPx1(-/-) cells. In contrast, the catalase inhibitor 3-aminotriazole (3AT) had no effect on CHP clearance. Application of CHP to wild type astrocytes was followed by a rapid and transient accumulation of GSSG, whereas in GPx1(-/-) cells no increase in the GSSG content was detected. Astrocytes from both mouse lines remained viable for up to 24 h following CHP exposure, however depletion of cellular GSH by pre-treatment with BSO compromised the viability of astrocytes, an effect that was stronger in GPx1(-/-) than in wild type cells. This cell death was almost completely prevented by iron chelators, whereas pre-incubation with iron increased CHP toxicity. These novel data demonstrate that the toxicity of organic hydroperoxides in astrocytes is iron-mediated, and that an intact GSH system is required for the effective removal of organic hydroperoxides and for protection from these peroxides.

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PubMed2005

Reactive oxygen species and the modulation of stroke.

Reactive oxygen species and oxidative state are slowly gaining acceptance in having a physiological relevance rather than just being the culprits in pathophysiological processes. The control of the redox environment of the cell provides for additional regulation in relation to critical cellular signal transduction pathways. Conversely, aberrant regulation of oxidative state manifesting as oxidative stress can predispose a cell to adverse outcome. The PI3-kinase/Akt pathway is one such pathway that is partially regulated via oxidative state and, in an oxidative stress paradigm such as ischemic reperfusion injury, may be inactivated, which can lead to potentiation and or exacerbation of cell death. Activation of NF(kappa)B has also been associated with oxidative stress. The role of NF(kappa)B in neuronal cell death is widely debated, with major studies highlighting both a pro- and an antiapoptotic role for NF(kappa)B with the outcome being region, stimulus, dose, and duration specific. This review hopes to make clear that oxidative state plays a key role in the regulation and control of numerous signal transduction pathways in the cell and that elucidating the mechanisms behind oxidative stress-mediated neuronal cell death is important in identifying potential putative targets for the treatment of neuropathologies such as stroke.

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PubMed2005

Diminished Akt phosphorylation in neurons lacking glutathione peroxidase-1 (Gpx1) leads to increased susceptibility to oxidative stress-induced cell death.

We have previously identified an increased susceptibility of glutathione peroxidase-1 (Gpx1)-/- mice to neuronal apoptosis following mid-cerebral artery (MCA) occlusion. This study was designed to elucidate the mechanisms involved in elevated neuronal cell death arising from an altered endogenous oxidant state. This was addressed in both an in vitro and in vivo model of oxidative stress in the form of exogenous H2O2 and cerebral ischaemia, respectively. Increased levels of cell death were detected in primary neurons lacking Gpx1 following the addition of exogenous H2O2. This increased apoptosis correlated with a down-regulation in the activation of the phospho-inositide 3-kinase [PI3K]-Akt survival pathway. The importance of this pathway in protecting against H2O2-induced cell death was highlighted by the increased susceptibility of wildtype neurons to apoptosis when treated with the PI3K inhibitor, LY294002. The Gpx1-/- mice also demonstrated elevated neuronal cell death following MCA occlusion. Although Akt phosphorylation was detected in the Gpx1-/- brains, activation was not seen in later reperfusion events, as demonstrated in wildtype brains. Previous studies have highlighted the importance of Akt phosphorylation in protecting against neuronal cell death following cerebral ischaemia-reperfusion. Our results suggest that the increased susceptibility of Gpx1-/- neurons to H2O2-induced apoptosis and neuronal cell death in vivo following cerebral ischaemia-reperfusion injury can be attributed in part to diminished activation of Akt. Perturbations in key anti-apoptotic mechanisms as a result of an altered redox state may have implications in the study of oxidative stress-mediated neuropathologies.

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PubMed2004

Akt phosphorylation and NFkappaB activation are counterregulated under conditions of oxidative stress.

This study was designed to elucidate the mechanisms involved in elevated cell death arising from an altered endogenous oxidant state. Increased levels of cell death were detected in cells lacking Gpx1 following the addition of exogenous H2O2. This increased apoptosis correlated with a down-regulation in the activation of the PI(3)K-Akt survival pathway. The importance of this pathway in protecting against H2O2-induced cell death was highlighted by the increased susceptibility of wild-type cells to apoptosis when treated with the PI(3)K inhibitor, LY294002. Activation of the oxidative stress sensitive transcription factor, NFkappaB, was elevated in the Gpx1-/- cells. Significantly, NFkappaB activation could be increased in wild-type cells through the addition of dominant-negative Akt. Therefore, our results suggest that the increased susceptibility of Gpx1-/- cells to H2O2-induced apoptosis can be attributed in part to diminished activation of Akt despite an up-regulation in the activation of the prosurvival NFkappaB. Thus, the PI(3)K-Akt and NFkappaB pathways can act independently of each other in an endogenous model of oxidative stress.

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PubMed2004

Fibroblasts derived from Gpx1 knockout mice display senescent-like features and are susceptible to H2O2-mediated cell death.

The Free Radical Theory of Aging proposes that reactive oxygen species (ROS) contribute to the pathophysiology of aging. Our previous data highlight the importance of antioxidant enzymes, superoxide dismutase 1 (Sod1) and glutathione peroxidase 1 (Gpx1), in regulating this process. Previously, we demonstrated that a perturbation in the Sod1-to-Gpx1 ratio, as a consequence of Sod1 overexpression, leads to senescence-like changes. We proposed that this was mediated via the Sod1 dismutation product H2O2, because H2O2 induced similar changes in control cells. However, it has been suggested that H2O2 production, via Sod1 dismutation, is rate-limited by the availability of the substrate O2*-, and therefore age-related changes may occur as a result of other functions of Sod1. In this study, we test this notion in fibroblasts derived from Gpx1 null mutant mice (Gpx1-/-) that have elevated H2O2 as a consequence of the lack of its removal by Gpx1. We demonstrate senescence-like changes in Gpx1-/- fibroblasts that include (1) reduced proliferative capacity, DNA synthesis, and responsiveness to EGF and serum; (2) elevated levels of Cip1; (3) increased NF-kappaB activation; and (4) morphological features of senescent cells. Gpx1-/- fibroblasts also demonstrate a dose-dependent susceptibility to H2O2-induced apoptosis. Our findings suggest that Gpx1 is protective against both ROS-mediated senescence-like changes and oxidant-mediated cell death.

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PubMed2004

Impact of oxidative stress on neuronal survival.

1. Reactive oxygen species and oxidative state are slowly gaining acceptance in having a physiological relevance rather than just being the culprits in pathophysiological processes. The control of the redox environment of the cell provides for additional regulation in relation to signal transduction pathways. Conversely, aberrant regulation of oxidative state manifesting as oxidative stress can predispose a cell to adverse outcome. 2. The phosphatidylinositol 3-kinase/akt pathway is one such pathway that is partially regulated via oxidative state and, in an oxidative stress paradigm such as ischaemic-reperfusion injury, may be inactivated, which can lead to exacerbation of cell death. 3. Activation of nuclear factor (NF)-kappaB has been associated with oxidative stress. The role of NF-kappaB in neuronal cell death is widely debated, with major studies highlighting both a pro- and anti-apoptotic role for NF-kappaB, with the outcome being region, stimulus, dose and duration specific. 4. Oxidative state plays a key role in the regulation and control of numerous signal transduction pathways in the cell. Elucidating the mechanisms behind oxidative stress-mediated neuronal cell death is important in identifying potential putative targets for the treatment of diseases such as stroke.

Animals↗

PubMed2004

Targeted disruption of SPI3/Serpinb6 does not result in developmental or growth defects, leukocyte dysfunction, or susceptibility to stroke.

Protease inhibitor 6 (PI-6/SERPINB6) is a widely expressed nucleocytoplasmic serpin. It inhibits granulocyte cathepsin G and neuronal neuropsin, and it is thought to protect cells from death caused by ectopic release or internalization of protease during stress such as infection or cerebral ischemia. To probe the biological functions of PI-6, we generated mice lacking its ortholog (SPI3/Serpinb6). SPI3-deficient mice developed normally and were fertile, and no abnormal pathology or increased sensitivity to cerebral ischemia was observed. There were no perturbations in leukocyte development or numbers, and recruitment of leukocytes to the peritoneal cavity was normal. SPI3-deficient mice were equally susceptible as wild-type mice to systemic Candida albicans infection, although there was a slight decrease in the ability of neutrophils from SPI3-deficient mice to kill C. albicans in vitro. Increased levels of a related inhibitor Serpinb1 (monocyte/neutrophil elastase inhibitor) in the tissues of targeted mice suggests that compensation by other serpins reduces the impact of SPI3 deficiency in these animals and may explain the lack of a more obvious phenotype.

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PubMed2003

Glutathione peroxidase-1 contributes to the neuroprotection seen in the superoxide dismutase-1 transgenic mouse in response to ischemia/reperfusion injury.

The authors hypothesized that glutathione peroxidase-1 (Gpx-1) contributes to the neuroprotection seen in the superoxide dismutase-1 transgenic (Sod-1 tg) mouse. To investigate this hypothesis, they crossed the Gpx-1 -/- mouse with the Sod-1 tg and subjected the cross to a mouse model of ischemia/reperfusion. Two hours of focal cerebral ischemia followed by 24 hours of reperfusion was induced via intraluminal suture. The Sod-1 tg/Gpx-1 -/- cross exhibited no neuroprotection when infarct volume was measured; indeed, infarct volume increased in the Sod-1 tg/Gpx-1 -/- cross compared with the wild-type mouse. Our results suggest that Gpx-1 plays an important regulatory role in the protection of neural cells in response to ischemia/reperfusion injury.

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PubMed2003

An imbalance in antioxidant defense affects cellular function: the pathophysiological consequences of a reduction in antioxidant defense in the glutathione peroxidase-1 (Gpx1) knockout mouse.

Aerobic cells are subjected to damaging reactive oxygen species (ROS) as a consequence of oxidative metabolism and/or exposure to environmental toxins. Antioxidants limit this damage, yet peroxidative events occur when oxidant stress increases. This arises due to increased radical formation or decreased antioxidative defenses. The two-step enzymatic antioxidant pathway limits damage to important biomolecules by neutralising superoxides to water. However, an imbalance in this pathway (increased first-step antioxidants relative to second-step antioxidants) has been proposed as etiological in numerous pathologies. This review presents evidence that a shift in favor of hydrogen peroxide and/or lipid peroxides has pathophysiological consequences. The involvement of antioxidant genes in the regulation of redox status, and ultimately cellular homeostasis, is explored in murine transgenic and knockout models. The investigations of Sod1 transgenic cell-lines and mice, as well as Gpx1 knockout mice (both models favor H(2)O(2) accumulation), are presented. Although in most instances accumulation of H(2)O(2) affects cellular function and leads to exacerbated pathology, this is not always the case. This review highlights those instances where, for example, increased Sod1 levels are beneficial, and indicates a role for superoxide radicals in pathogenesis. Studies of Gpx1 knockout mice (an important second-step antioxidant) lead us to conclude that Gpx1 functions as the primary protection against acute oxidative stress, particularly in neuropathological situations such as stroke and cold-induced head trauma, where high levels of ROS occur during reperfusion or in response to injury. In summary, these studies clearly highlight the importance of limiting ROS-induced cellular damage by maintaining a balanced enzymatic antioxidant pathway.

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PubMed2002

A mouse model of spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease, caused by the expansion of a trinucleotide repeat (TNR) in exon 1 of the androgen receptor (AR) gene. This disorder is characterized by degeneration of motor and sensory neurons, proximal muscular atrophy, and endocrine abnormalities, such as gynecomastia and reduced fertility. We describe the development of a transgenic model of SBMA expressing a full-length human AR (hAR) cDNA carrying 65 (AR(65)) or 120 CAG repeats (AR(120)), with widespread expression driven by the cytomegalovirus promoter. Mice carrying the AR(120) transgene displayed behavioral and motor dysfunction, while mice carrying 65 CAG repeats showed a mild phenotype. Progressive muscle weakness and atrophy was observed in AR(120) mice and was associated with the loss of alpha-motor neurons in the spinal cord. There was no evidence of neurodegeneration in other brain structures. Motor dysfunction was observed in both male and female animals, showing that in SBMA the polyglutamine repeat expansion causes a dominant gain-of-function mutation in the AR. The male mice displayed a progressive reduction in sperm production consistent with testis defects reported in human patients. These mice represent the first model to reproduce the key features of SBMA, making them a useful resource for characterizing disease progression, and for testing therapeutic strategies for both polyglutamine and motor neuron diseases.

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PubMed2002

Mice lacking glutathione peroxidase-1 activity show increased TUNEL staining and an accelerated inflammatory response in brain following a cold-induced injury.

The mechanisms leading to neurodegeneration are complex and multifactorial. Oxidative stress has been identified as an important constituent in this process and the use of transgenic and knockout mice has allowed the role of key components of the antioxidant pathway to be evaluated. In this study, we have used mice lacking the glutathione peroxidase-1 gene in order to determine the consequences of a reduced capacity to neutralize hydrogen peroxide toward the pathological outcomes following cold-induced brain injury. Analysis of brain cryosections using TUNEL staining revealed a significant increase in brain cell death in knockout mice compared to that seen in wild-type mice. Interestingly, cell death appeared to be uncoupled to a neuro-inflammatory response which was observed in both knockout and wild-type mice but which proceeded in an accelerated manner in glutathione peroxidase-1 knockout mice at 24 h, rapidly diminishing by 96 h postinjury. Our data suggest an important role for glutathione peroxidase-1 in modulating molecular pathways involved in both the level of cell death and inflammatory cascades in brain through its antioxidant capacity in regulating levels of oxygen species such as hydrogen peroxide.

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