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Stephen R Robinson

Publications and source records attributed to Stephen R Robinson.

At least 19 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.

Animals↗
PubMed2006

Morphology, characterization, and distribution of retinal photoreceptors in the Australian lungfish Neoceratodus forsteri (Krefft, 1870).

The Australian lungfish Neoceratodus forsteri (Dipnoi) is an ancient fish that has a unique phylogenetic relationship among the basal Sarcopterygii. Here we examine the ultrastructure, histochemistry, and distribution of the retinal photoreceptors using a combination of light and electron microscopy in order to determine the characteristics of the photoreceptor layer in this living fossil. Similar proportions of rods (53%) and cones (47%) reveal that N. forsteri optimizes both scotopic and photopic sensitivity according to its visual demands. Scotopic sensitivity is optimized by a tapetum lucidum and extremely large rods (18.62 +/- 2.68 microm ellipsoid diameter). Photopic sensitivity is optimized with a theoretical spatial resolving power of 3.28 +/- 0.66 cycles degree(-1), which is based on the spacing of at least three different cone types: a red cone containing a red oil droplet, a yellow cone containing a yellow ellipsoidal pigment, and a colorless cone containing multiple clear oil droplets. Topographic analysis reveals a heterogeneous distribution of all photoreceptor types, with peak cone densities predominantly found in temporal retina (6,020 rods mm(-2), 4,670 red cones mm(-2), 900 yellow cones mm(-2), and 320 colorless cones mm(-2)), but ontogenetic changes in distribution are revealed. Spatial resolving power and the diameter of all photoreceptor types (except yellow cones) increases linearly with growth. The presence of at least three morphological types of cones provides the potential for color vision, which could play a role in the clearer waters of its freshwater environment.

Animals↗
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.

Animals↗
PubMed2004

Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells.

The ferrozine-based colorimetric assay described here permits the quantitation of iron in cultured cells in amounts ranging between 0.2 and 30 nmol. Ferrous and ferric iron were detected equally well by the assay and the accuracy was unaffected by other divalent metal cations. This colorimetric assay was used to study iron accumulation in brain astrocytes that had been cultured in 24-well dishes. Iron complexed to cellular proteins was made accessible to ferrozine by treatment of cell lysates with acidic KMnO(4) solution. The basal amounts of iron in untreated astrocyte cultures were approximately 10 nmol iron per mg protein. Incubation of the cells with ferric ammonium citrate caused the total cellular iron content to increase in a concentration-dependent manner. The estimates of cellular iron content that were obtained with the ferrozine-based assay did not differ from those determined by atomic absorption spectroscopy. The colorimetric assay described here provides a sensitive, cheap, and reliable method for the quantitation of intracellular iron and for the investigation of iron accumulation in cultured cells.

Animals↗
PubMed2004

Endogenous glutathione and catalase protect cultured rat astrocytes from the iron-mediated toxicity of hydrogen peroxide.

Primary astrocyte cultures from rat brain were exposed to hydrogen peroxide (H2O2) to investigate peroxide toxicity and clearance by astrocytes. After bolus application of H2O2 (100 microM), the peroxide was eliminated from the incubation medium following first-order kinetics with a half-time of approximately 4 min. The rate of peroxide detoxification was significantly slowed by pre-incubating the cells with the glutathione synthesis inhibitor buthionine sulfoximine (BSO), or the catalase inhibitor 3-amino-1,2,4-triazole (3AT), and was retarded further when both treatments were combined. H2O2 application killed a small proportion of cells, as indicated by the levels of the cytosolic enzyme lactate dehydrogenase in the media 1 and 24h later. In contrast, cell viability was strongly compromised when the cells were pre-incubated with 3AT and/or BSO before peroxide application. The iron chelator deferoxamine completely prevented this cell loss. These results demonstrate that chelatable iron is involved in the toxicity of H2O2 and that both the glutathione system and catalase protect astrocytes from this toxicity.

Animals↗
PubMed2004

Pharmacological but not physiological concentrations of melatonin reduce iron-induced neuronal death in rat cerebral cortex.

Iron-mediated oxidative stress occurs in a wide variety of neurological disorders. The present study has investigated whether melatonin can alter the proportion of neurons that die in the 24 h period following 1.0 microl intracortical injections of 1.0 mM ferric ammonium citrate (FAC) or 0.9% saline. Rats which received systemic infusions of melatonin (5 mg/kg body weight per day) displayed a 40% reduction (P = 0.019) in the proportion of neurons killed by FAC. By contrast, reduction of endogenous melatonin by continuous light exposure did not significantly affect the extent of neuronal death. Furthermore, elevated or reduced melatonin levels did not alter the number of neurons killed by saline injections. We conclude that pharmacological concentrations of melatonin protect neurons against iron-induced injury.

Animals↗
PubMed2004

Iron accumulation, iron-mediated toxicity and altered levels of ferritin and transferrin receptor in cultured astrocytes during incubation with ferric ammonium citrate.

The cellular uptake and storage of iron have to be tightly regulated in order to provide iron for essential cellular functions while preventing the iron-catalysed generation of reactive oxygen species (ROS). In contrast to cells in other organs, little is known about the regulation of iron metabolism in brain cells, particularly in astrocytes. To investigate the regulation of iron metabolism in astrocytes we have used primary astrocyte cultures from the brains of newborn rats. After application of ferric ammonium citrate (FAC), cultured astrocytes accumulated iron in a time- (0-48 h) and concentration-dependent (0.01-1 mm) manner. This accumulation was prevented if FAC was applied in combination with the iron-chelator deferoxamine (DFX). Application of FAC to astrocyte cultures caused a strong increase in the cellular content of the iron storage protein ferritin and a decrease in the amount of transferrin receptor (TfR), which is involved in the transferrin-mediated uptake of iron into cells. In contrast, application of DFX strongly increased the level of TfR. Both up-regulation of ferritin content by iron application and up-regulation of TfR content by DFX were prevented by the protein synthesis inhibitor cycloheximide (CHX). During incubation of astrocytes with FAC, a mild and transient increase in the extracellular activity of the cytosolic enzyme lactate dehydrogenase and in the concentration of intracellular ROS was observed. In contrast, prevention of protein synthesis by CHX during incubation with FAC resulted in significantly more cell loss and a persistent and intense increase in the production of intracellular ROS. These results demonstrate that both iron accumulation and deprivation modulate the synthesis of ferritin and TfR in astrocytes and that protein synthesis is required to prevent iron-mediated toxicity in astrocytes.

Animals↗
PubMed2004

The amyloid paradox: amyloid-beta-metal complexes can be neurotoxic and neuroprotective.

Senile plaques in the brains of people with Alzheimer disease (AD) are primarily composed of the amyloid-beta (Abeta) peptide and contain substantially elevated levels of iron, copper and zinc. These metals bind to Abeta and have been reported to increase the toxicity of Abeta to cultured neurones. Other reports have demonstrated that Abeta can reduce the neurotoxicity of metal ions, suggesting that the interaction can, under some circumstances, be protective. To investigate these apparently conflicting results, human Abeta1-42 was co-injected with iron, copper or zinc (at the concentrations found in plaques) into rat cerebral cortex, and the resulting numbers of dying neurones were compared. It was found that Abeta complexed with either iron or zinc was more toxic than Abeta alone. In contrast, Abeta-copper complexes were not neurotoxic. Surprisingly, we observed that when iron or copper were combined with Abeta, the neurotoxicity of these metals was substantially reduced, suggesting that Abeta may help to limit the toxicity of redox-active metal ions, thereby assisting the antioxidant defence of the brain. Thus paradoxical effects occur when Abeta complexes with metal ions, where Abeta-metal complexes are capable of being neurotoxic and neuroprotective.

Amyloid beta-Peptides↗
PubMed2004

Physiological roles of amyloid-beta and implications for its removal in Alzheimer's disease.

The underlying pathological cause of Alzheimer's disease has been postulated to be an excess of amyloid-beta (Abeta) which aggregates into toxic fibrillar deposits within the extracellular space of the brain, thereby disrupting neuronal and synaptic function and eventually leading to neuronal degeneration and dementia. As a result, therapeutic strategies have been developed that are designed to remove Abeta from the brain. Caution needs to be exercised concerning such strategies because, in addition to its presence in neuritic plaques, Abeta has a widespread distribution through the brain and body, even in cognitively normal individuals. Evidence indicates that instead of being a toxic peptide, soluble Abeta serves a variety of physiological functions, including modulation of synaptic function, facilitation of neuronal growth and survival, protection against oxidative stress, and surveillance against neuroactive compounds, toxins and pathogens. These physiological functions must be taken into account when strategies are developed to reduce Abeta load in Alzheimer's disease. Ideally, such strategies should target forms of Abeta that are not bioavailable, such as fibrillar Abeta, or forms that are regarded to be overexpressed in Alzheimer's disease (such as oligomers) while leaving normal soluble Abeta1-40 and Abeta1-42 intact. At present none of the available therapeutic strategies appears to have such selectivity. Until these technical limitations and the uncertainties regarding the effect of depletion of Abeta from the brain are resolved, it would not be prudent to begin further clinical trials.

Alzheimer Disease↗
PubMed2003

Human Abeta1-42 reduces iron-induced toxicity in rat cerebral cortex.

Senile plaques, the major neuropathological lesions of Alzheimer's disease (AD), are composed primarily of amyloid-beta (Abeta) peptide and contain high concentrations of iron (1.0 mM). We have previously shown that intracortical injections of 1.0 mM iron to adult rats produce significantly more neuronal loss than control injections of saline vehicle, whereas injections of Abeta do not. Because iron has been shown to increase the in vitro toxicity of Abeta, the present study was undertaken to determine whether iron can make Abeta neurotoxic in vivo. Abeta and 1.0 mM iron (as ferric ammonium citrate) were coinjected into rat cerebral cortex, and the neuronal loss was compared with that produced by pure Abeta or pure iron. The human and rat variants of Abeta(1-42) were compared to determine whether they produce the same amount of neuronal loss when combined with iron. Coinjection of iron with either Abeta variant caused significantly more neuronal loss than Abeta peptide alone, suggesting that iron may contribute to the toxicity associated with senile plaques. Rat Abeta(1-42) combined with iron was as toxic as iron alone, whereas iron combined with human Abeta(1-42) was significantly less toxic. This latter finding indicates that fibrillar human Abeta is able to reduce iron-induced neurotoxicity in vivo and raises the interesting possibility that senile plaques in AD may represent a neuroprotective response to the presence of elevated metal ions.

Alzheimer Disease↗
PubMed2003

Alzheimer vaccine: amyloid-beta on trial.

A new therapeutic approach is being developed for the treatment of Alzheimer's disease (AD). This approach involves the deliberate induction of an autoimmune response to amyloid-beta (Abeta) peptide, the constituent of neuritic plaques that is thought to cause the neurodegeneration and dementia in AD. If this approach is to be effective, antibodies must be produced that can selectively target the toxic forms of Abeta, while leaving the functionally-relevant forms of Abeta and its precursor protein untouched. Furthermore, an approach needs to be found that avoids provoking an acute neuroinflammatory response. The situation is made even more challenging by uncertainty regarding which isoforms of Abeta contribute to the pathogenesis of AD.

Alzheimer Disease↗
PubMed2002

Iron: a pathological mediator of Alzheimer disease?

Brains from patients with Alzheimer disease (AD) show a disruption in the metabolism of iron, such that there is an accumulation of iron in senile plaques, and an altered distribution of iron transport and storage proteins. One of the earliest events in AD is the generation of oxidative stress, which may be related to the generation of free radicals by the excess iron that is observed in the disease. Iron has also been shown to mediate the in vitro toxicity of amyloid-beta peptide, and the presence of iron in most in vitro systems could underlie the toxicity that is normally attributed to amyloid-beta in these studies. In contrast, several recent studies have suggested that amyloid-beta may decrease oxidative stress and decrease the toxicity of iron. Continued examination of the complex interactions that occur between iron and amyloid-beta may assist in the elucidation of the mechanisms that underlie the neurodegeneration that leads to dementia in AD.

Alzheimer Disease↗
PubMedDate not supplied

Lessons from the AN 1792 Alzheimer vaccine: lest we forget.

Recent clinical and neuropathological data show that the AN 1792 vaccine enhanced the production of Abeta antibodies in the sera of Alzheimer's disease (AD) patients, but it appears to have been ineffective at stimulating the removal of Abeta deposits from the brain or at slowing the rate of cognitive decline. The 19 cases of meningoencephalitis were not linked in an obvious way to serum antibody titre, but they may have been linked to infiltration of the brain by antibodies and/or T-cells. Brain imaging indicated that oedema associated with the neuroinflammation did not reflect the typical distribution of neuritic plaques in AD. These outcomes were not anticipated by experiments on transgenic mice because compared to humans, these mice have less genetic variability, and their plaques have a different chemical composition, making them far more soluble and easier to remove. Furthermore, the consequences of vaccination are different. Vaccination of transgenic mice removes superfluous human Abeta while leaving endogenous mouse Abeta intact, whereas in humans the immune response is directed against an endogenous target that occurs naturally and is present in healthy brain tissue. The most important lesson to be learned from the AN 1792 trials is that new strategies for treating AD should not be tested on humans until they have been extensively tested on non-murine species.

Alzheimer Disease↗
PubMedDate not supplied

Challenges and directions for the pathogen hypothesis of Alzheimer's disease.

This paper critically reviews the possibility that infiltration of the brain by pathogens (e.g. Herpes simplex virus type 1 (HSV1) or Chlamydophila pneumoniae (Cp)) acts as a trigger or co-factor for Alzheimer's disease (AD). The evidence currently available is limited and in some cases inconsistent, but it does justify the need for more vigorous investigation of this hypothesis. An issue of particular concern is the paucity of experimental evidence showing that pathogens can elicit the neuropathological changes and cognitive deficits that characterise AD. Other weaknesses include a failure to obtain independent confirmation of Cp in AD brains, and a lack of evidence for HSV1 proteins or intact virions in AD brain tissue. Future avenues of investigation that might prove fruitful include epidemiological investigations of the incidence of AD in individuals who are either immunosuppressed or have received chronic antiviral or antibiotic therapy. There is also a need to consider systemic infections as potential contributors to the pathogenesis of AD.

Alzheimer Disease↗
PubMedDate not supplied

Abeta as a bioflocculant: implications for the amyloid hypothesis of Alzheimer's disease.

Research into Alzheimer's disease (AD) has been guided by the view that deposits of fibrillar amyloid-beta peptide (Abeta) are neurotoxic and are largely responsible for the neurodegeneration that accompanies the disease. This 'amyloid hypothesis' has claimed support from a wide range of molecular, genetic and animal studies. We critically review these observations and highlight inconsistencies between the predictions of the amyloid hypothesis and the published data. We show that the data provide equal support for a 'bioflocculant hypothesis', which posits that Abeta is normally produced to bind neurotoxic solutes (such as metal ions), while the precipitation of Abeta into plaques may be an efficient means of presenting these toxins to phagocytes. We conclude that if the deposition of Abeta represents a physiological response to injury then therapeutic treatments aimed at reducing the availability of Abeta may hasten the disease process and associated cognitive decline in AD.

Alzheimer Disease↗