• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Eur J Neurosci. Apr 2009; 29(7): 1335–1347.
PMCID: PMC2777252

Fenton chemistry and oxidative stress mediate the toxicity of the β-amyloid peptide in a Drosophila model of Alzheimer’s disease

Abstract

The mechanism by which aggregates of the β-amyloid peptide (Aβ) mediate their toxicity is uncertain. We show here that the expression of the 42-amino-acid isoform of Aβ (Aβ1–42) changes the expression of genes involved in oxidative stress in a Drosophila model of Alzheimer’s disease. A subsequent genetic screen confirmed the importance of oxidative stress and a molecular dissection of the steps in the cellular metabolism of reactive oxygen species revealed that the iron-binding protein ferritin and the H2O2 scavenger catalase are the most potent suppressors of the toxicity of wild-type and Arctic (E22G) Aβ1–42. Likewise, treatment with the iron-binding compound clioquinol increased the lifespan of flies expressing Arctic Aβ1–42. The effect of iron appears to be mediated by oxidative stress as ferritin heavy chain co-expression reduced carbonyl levels in Aβ1–42 flies by 65% and restored the survival and locomotion function to normal. This was achieved despite the presence of elevated levels of the Aβ1–42. Taken together, our data show that oxidative stress, probably mediated by the hydroxyl radical and generated by the Fenton reaction, is essential for Aβ1–42 toxicity in vivo and provide strong support for Alzheimer’s disease therapies based on metal chelation.

Keywords: aggregation, chelation therapy, hydroxy radical, iron

Introduction

There is a growing consensus that smaller, soluble aggregates of the β-amyloid peptide (Aβ), rather than mature amyloid plaques, are the pathogenic species in Alzheimer’s disease (AD) (Lambert et al., 1998; Walsh et al., 2002). However, the mechanism by which these aggregates mediate their toxicity remains unclear. As Aβ is generated in the extracellular space, or more likely within the lumen of endocytic vesicles (Koo & Squazzo, 1994; Refolo et al., 1995), its toxic effects may be mediated by membrane damage or by interactions with membrane-bound proteins. There is evidence that Aβ aggregates can degrade the electrical resistance of membranes (Kayed et al., 2004; Demuro et al., 2005), possibly by forming pores (Lashuel et al., 2002), or alternatively they may interact with membrane receptors or even gain access to the cytoplasm. Membranes can also be damaged by the reactive oxygen species that are generated by Aβ aggregates in the presence of metals such as copper, zinc or iron (Bush, 2003). Subsequent pathological processes include mitochondrial damage (Abramov et al., 2004), tau phosphorylation with consequent axonal transport dysfunction and the initiation of cell death (Kienlen-Campard et al., 2002; Wei et al., 2002; Jo et al., 2004). However, until recently (Cao et al., 2008) it has been impossible to take a global view to ask which biological processes are essential for the development of the disease and which are downstream consequences of neurotoxicity. Knowing which biological processes are directly involved in initiating AD will allow us to focus on those upstream targets that have the greatest therapeutic potential.

We have developed a model of AD that is based on the expression of the human Aβ in fly neurons by coupling it to an N-terminal secretion signal peptide (Crowther et al., 2005). The Aβ1–42 but not the Aβ1–40 control accumulates in the brain and results in decreased lifespan and impaired locomotor performance. These phenotypes are more marked in flies expressing the E22G (Arctic) mutant of the Aβ1–42, which causes increased aggregation of Aβ and is responsible for early onset familial AD (Nilsberth et al., 2001). Here, we use our Drosophila model of AD to identify the pathways and intermediates that are critical for Aβ-mediated toxicity in vivo.

Materials and methods

Drosophila stocks

The following stocks were generous gifts: UAS-CAT [catalase (CAT) upstream activating sequence (UAS) inducible transgene] (Anderson et al., 2005), UAS-SOD1 [CuZn-superoxide dismutase (SOD)1] (Anderson et al., 2005), UAS-SOD1-IR (RNAi line for SOD1) (Missirlis et al., 2003) and UAS-mitSOD2 (Mn-SOD2) (Anderson et al., 2005) from Professor John Phillips (Guelph, Canada), UAS-Sniffer (carbonyl reductase) (Botella et al., 2004) from Professor Stephan Schneuwly (Regensburg, Germany) and UAS-GST [glutathione-S-transferase (GST) S1] (Whitworth et al., 2005) from Dr Alex Whitworth (Sheffield, UK). We used previously characterized UAS-CAT, UAS-SOD1 and UAS-mitSOD2 stocks (Anderson et al., 2005) as they increase enzymatic activity by up to 200%. Stocks of mutant SOD1n108 (Phillips et al., 1995) and elavc155-GAL4 were from the Bloomington stock centre (Indiana, USA). Flies carrying Aβ transgenes [Aβ1–40 (Alz40.1), Aβ1–42 (Alz42.2 and Alz42.3) or Arctic1–42 (AlzArc1)] have been described previously (Crowther et al., 2005; Luheshi et al., 2007). The transgenes are each representative of six independent transgenic lines and each transgene drives similar levels of mRNA whether alone or in combination (such as Alz42.2 + Alz42.3).

DNA constructs

The cDNA for Drosophila ferritin 1 heavy chain (Fer1HC) was isolated from EST clone GH24060 (Berkley Drosophila Genome Project). This cDNA lacked the iron response element resulting in the expression of a constitutively active form of Fer1HC. The cDNA for Drosophila ferritin 2 light chain (Fer2LC) naturally lacks an iron response element and was isolated from the EST clone AT16780 (Berkley Drosophila Genome Project). Both cDNAs were inserted downstream of GAL4 UASs (UAS-Fer1HC and UAS-Fer2LC) in the pUAST plasmid by directional cloning following EcoRI and XhoI digestion.

Affymetrix cDNA microarray

Individual samples were each hybridized to a Drosophila Genome GeneChip® Array following standard Affymetrix protocols. Affymetrix Microarray Suite 5 was used to generate signal values and detection calls. Probe level intensity data were adjusted for background, normalized and log transformed using the robust multichip average pre-processing method (Irizarry et al., 2003) using Rosetta Resolver® 7.1.

Ratio data for each individual probe were created as follows. Aβ1–40 baseline samples were created for each time-point by pooling all replicates (see Supplementary material, Fig. S1a). The individual replicates for each test condition (Arctic Aβ1–42 and Aβ1–42) were then compared with the age-matched control Aβ1–40 pool. All statistical analyses were performed on log10 ratio data.

Gene expression differences were determined by one-way, error-weighted anova on ratio data by comparing Aβ1–42 and Arctic mutants with age-matched Aβ1–40 controls (factor = genotype, n = 4 per group). Genes were only considered in the analysis of over-represented biological themes if the differential expression was highly significant (P<0.01).

Characterization of over-represented biological themes

Gene Ontology (Ashburner et al., 2000) enrichment analysis was performed using the Gene Ontology tree machine (http://bioinfo.vanderbilt.edu/gotm/) (Zhang et al., 2004). In summary, a hypergeometric test was performed to determine whether a particular biological process or molecular function was disproportionately represented in the set of genes that were differentially expressed as compared with all of the genes in the GeneChip® array. Where n= number of genes that are differentially expressed between two experimental conditions (e.g. between Arctic Aβ1–42 and Aβ1–40 on day 0), N= total number of genes on the array, K= number of genes on the array that belong to the category of interest and k= number of genes that belong to the category that are differentially expressed, the significance (P) of enrichment for a given category is determined by

equation image

A ratio of enrichment, R, is calculated as R= k/ke where ke = (n/N)K is the expected value for a given category if n = number of genes were a random sample of uniformly selected genes from the reference set of all genes on the chip.

Gene Search element screen

A library of 3000 unique insertions of the Gene Search (GS) element was generated by mobilizing the GS element from the X chromosome (DGRC number 200079) to the autosomes by crossing with flies expressing the Delta2-3 transposase. The first eclosed fly with a stably-jumped GS element from each mobilization cross was used to establish a GS line. Stocks were maintained by monitoring eye colour. The effect of the Gal-4 activated GS element on the longevity of flies expressing the Arctic mutant of Aβ1–42 was determined by crossing male flies with elavc155-Gal4 (elav-Gal4) on the X chromosome and the Arctic1–42 transgene on the second chromosome with virgin females with a floating GS element. All of the female offspring had both elav-Gal4 and the Arctic1–42 transgene but only half had the GS element; the unmodified population (without the GS element) provided an internal control for each longevity assay. The null hypothesis was that the presence of the GS element made no difference to the longevity of the flies expressing the Arctic1–42 transgene.

Longevity assays for the primary screen were performed at 25°C, blind to the identity of the GS elements. Live flies were counted and the food changed on days 1, 3 and 5 of a 7 day cycle. A mean number of 17 flies was assessed per GS line (total number of flies assessed, 50 320; minimum number of flies assessed per GS line, 10). To detect suppression of the longevity phenotype we determined the time to 75% death, which is the median survival of the flies in which the GS element has prolonged their life. These survival times were normally distributed (n = 2893, mean 21.6 days, SD 3.8 days) and any lifespan that was more than two SDs greater than the mean was defined as significant.

In the secondary screen the chromosomal site of insertion was determined by classical genetics and homozygous GS flies were crossed so that all flies in the longevity assays expressed the activated GS element. This secondary screen used homozygous GS element stocks and assessed more than 30 flies, permitting robust comparison of the survival of GS-modified and unmodified Arctic1–42 flies. The site of the GS element insertion was identified in those lines with confirmed modifier activity by inverse polymerase chain reaction using the FlyChip facility (http://www.drosdel.org.uk/molecular_methods.php). The molecular function of genes with inserts within the coding sequence was determined from FlyBase. For inserts in non-coding DNA, the molecular function was determined for the genes on either side of the insert. The effect of the activated GS element on the survival of control flies and flies expressing the AlzArc1 transgene was also determined. GS lines in which the modification of the Aβ longevity phenotype was not confirmed or in which the GS element had a marked non-specific effect on control flies were discarded.

Longevity assays for the secondary screen and genetic modifier assessment

Longevity assays in the secondary screen and for each of the subsequent genetic modifiers followed at least 80 flies per genotype in groups of 10 flies per vial. Live flies were counted and their food changed on days 1, 3 and 5 of a 7 day cycle. For assessing the efficacy of metal chelation, clioquinol (Calbiochem) was dissolved in dimethylsulphoxide and the solution added to fly food to give a final concentration of 0.2% v/v. Survival curves were plotted using the Kaplan–Meier estimator. The statistical significance was calculated using the log rank test within the spss 11.0 statistical package. The null hypothesis in all of the longevity assays was that the presence of the GS element made no difference to the longevity of the flies expressing the Arctic1–42 transgene.

Assay of sensorimotor performance

The sensorimotor performance of the flies was determined using a previously described negative geotaxis assay (Rival et al., 2004). Fifteen flies were placed in a sterile plastic column (25 cm tall × 1.5 cm internal diameter) and tapped to the bottom. After 45 s the flies at the top of the column (Ntop) and the flies remaining at the bottom (Nbot) were counted. Three trials were performed at 1 min intervals. The performance index was defined as (15 + NtopNbot)/30. Statistical analysis was performed using the two-tailed Student’s t-test.

Quantitation of β-amyloid peptides

Drosophila were cultured at 25°C for 5 days after eclosion and then five heads were homogenized in 50 μL of 5 m guanidinium HCl, 5 mm EDTA and 50 mm hepes, pH 7.3. Following centrifugation for 5 min at 12 000 g, 20 μL of the clear supernatant was removed and mixed with 180 μL of 25 mm HEPES, pH 7.3, 1 mm EDTA and 0.1% w/v bovine serum albumin with protease inhibitors (Complete™, Roche). Triplicate 25 μL aliquots were mixed with an equal volume of phosphate-buffered saline containing 2% w/v bovine serum albumin, 0.2% v/v Tween-20 and protease inhibitors (Complete™, Roche) in wells on a MESO microtitre plate (Standard bind, MA6000, no. P11AA-1; Meso Scale Discovery, MD, USA). The reaction was started by adding 25 μL of 4 μg/mL solutions of biotinylated 6E10 or 4G8 (Signet Laboratories, MA, USA) monoclonal antibodies. After mixing a further 25 μL aliquot of 1 μg/mL Ruthenium-labelled G2-10 or G2-11 (The Genetics Company, Switzerland), monoclonal antibody solution was added to each well. Following an overnight incubation at 25°C, the plates were washed twice with phosphate-buffered saline, 150 μL of S Read Buffer (R92SC-1, Meso Scale Discovery, MD, USA) was added and the measurement was taken in a Sector PR instrument (Meso Scale Discovery). Statistical analysis was performed using the two-tailed Student’s t-test.

Hydrogen peroxide sensitivity assay

Two-day-old flies were cultured in a vial containing only filter paper soaked in an aqueous solution of 2% w/v sucrose and 10% v/v H2O2 or a control solution of 2% w/v sucrose alone. Ten vials of 15 flies were cultured for each genotype at 25°C with the soaked paper being replaced twice per day. Statistical analysis was performed using the two-tailed Student’s t-test.

Carbonyl assay

Five adult flies (28 days old, grown at 25°C) were decapitated and the heads were immediately homogenized in water, sonicated and centrifuged at 12 000 g for 10 min and the clear supernatant assayed for protein content using the Bradford method. The protein concentration was adjusted to 5 μg/mL by the addition of phosphate-buffered saline. Protein carbonyl groups were assayed using an enzyme-linked immunosorbent assay-based protocol described by Alamdari et al. (2005). Protein carbonyl groups were reacted with dinitrophenol hydrazine (Sigma-Aldrich) and the resulting dinitrophenol adducts were detected using an anti-DNP rabbit polyclonal antibody (Sigma-Aldrich). Statistical analysis was performed using the two-tailed Student’s t-test.

Iron and zinc determination in fly head extracts by mass spectrometry

Flies were cultured at 29°C and decapitated at 10 days old. Triplicates of 20 fly heads per condition were weighed by difference into a new 15 mL screw-top polypropylene centrifuge tube (part no. 2086-500, Elkay, UK). Nitric acid (500 μL) (‘Trace Select’, part no. 84385, Fluka, UK) was added and the tubes were sealed and incubated overnight at 65°C such that there was no visible solid matter remaining. The sample was prepared by resuspending the extracts in 5 mL of double-distilled water before analysis using an Elan 6100 DRC ICP/MS (Perkin-Elmer). The reaction cell was used for all measurements with the following operating conditions: 1.04 L/min nebulizer flow, 1100 W radio frequency power and a dynamic reaction cell gas flow of 0.7 mL/min of ammonia. Iron (Mr 55.9349), zinc (Mr 65.9260) and calcium (Mr 43.9555), but not copper (Mr 62.9298), were reliably detected. As calcium is not chelated by clioquinol it was used to control for the quantity of tissue in each sample.

Results

Microarray analysis of gene expression in flies expressing β-amyloid peptide supports the importance of oxidative stress in β-amyloid peptide toxicity

Affymetrix microarrays were used to identify gene expression signatures (one-way anova, P < 0.001) for flies expressing wild-type Aβ1–42 at day 0, 3 and 8 of adult life and Arctic1–42 at day 0 and 3 by comparison to age-matched Aβ1–40 controls. The derived gene expression signatures for flies expressing wild-type and Arctic1–42 were then used to determine the biological processes, molecular functions and cellular components that were over-represented in each set (in supplementary Fig. S1b). The role of redox genes appeared most apparent in the early stages of Aβ1–42 expression (Fig. 1); this is most clearly seen for flies expressing wild-type Aβ1–42 at day 3 (top part, shaded) where these genes comprised six of the 10 families of enriched genes. Genes that were classified as having ‘oxidoreductase activity’ were significantly enriched at both day 3 and 8 in flies expressing wild-type Aβ1–42 (P < 0.01). Similarly, carbonyl reductase (NADPH) activity was enriched in the day 3 signature of flies expressing Arctic1–42 (P < 0.01). Taken together, these observations provide strong support for the role of oxidative stress-related genes in the response of the fly brain to the expression of the Aβ.

Fig. 1
The expression signature of genes for flies expressing Aβ1–42 at day 3 (top part) and day 8 (bottom part) of adult life was determined and analysed for over-representation of genes belonging to particular molecular functions. Only functional ...

P-element screen for modifiers of β-amyloid peptide toxicity implicate redox regulation as an important target of modifier activity

A 3000-line library of unique GS (Toba et al., 1999) element inserts was screened for lines that modified the longevity of Drosophila expressing Aβ1–42 in their central nervous system. The GS elements can either disrupt gene function when they insert within essential coding or non-coding DNA, or else they can bidirectionally upregulate neighbouring genes. In our screen 1.5% of the GS inserts resulted in an increase in median survival that was more than two SDs away from the mean, whereas 0.5% of the inserts significantly reduced survival. A secondary longevity assay was performed to confirm the initial findings but also to determine the effect of the GS elements on the survival of control flies that did not express Aβ. The insertions that specifically modified the longevity of Aβ-expressing flies, and not controls, were classified into 18 suppressor and three enhancer groups according to the identity of the neighbouring genes. Seven of these 21 classes were adjacent to genes with a predictable role in oxidative stress (Table 1 and Fig. 2). The selected suppressor GS elements increased median survival by 25–96% in flies expressing Aβ but had no, or little, effect in control flies (Fig. 2, filled and empty bars, respectively).

Table 1
GS elements inserted within or adjacent to genes involved in oxidative stress modify the toxicity of Arctic
Fig. 2
The percentage increase in median survival in flies expressing Arctic1–42 that was attributable to the GS element was calculated (filled bars). Control flies (n = 100) possessing elav-Gal4±the GS element but lacking the Arctic ...

Flies expressing β-amyloid peptide1–42 are more sensitive to oxidative stress and have higher levels of oxidative damage

The genetic screen and microarray data implicated oxidative stress as playing a central role in the toxicity of the Aβ1–42. This was assessed by testing the response of flies to an oxidative insult in the form of H2O2 in their food. In these experiments we observed that flies expressing wild-type Aβ1–42 were more likely to die if their food was supplemented with 10% v/v H2O2 than either control flies (Fig. 3a, elav-Gal4 w1118 vs. elav-Gal4 UAS-Aβ1–42, P < 0.05) or flies expressing Aβ1–40 (Fig. 3a, elav-Gal4 UAS-Aβ1–40 vs. elav-Gal4 UAS-Aβ1–42, P < 0.05). When we quantified oxidative damage by measuring the levels of carbonyl groups in protein extracts of fly heads we found that flies expressing Aβ1–40 had carbonyl levels that were similar to those of control flies (Fig. 3b, elav-Gal4 w1118 vs. elav-Gal4 UAS-Aβ1–40), whereas flies expressing a single Aβ1–42 transgene had almost double the carbonyl levels of flies expressing Aβ1–40 (Fig. 3b, elav-Gal4 UAS-Aβ1–42 vs. elav-Gal4 UAS-Aβ1–40, P < 0.05).

Fig. 3
Flies expressing Aβ1–42 are more sensitive to oxidative stress and have higher levels of oxidative damage. Supplementing the fly food with 10% v/v H2O2 for 72:00 h (a, shaded bars) was specifically toxic to flies expressing Aβ ...

Transgenic over-expression of single antioxidative stress genes rescues the β-amyloid peptide-induced longevity phenotype

The ability of antioxidative stress genes identified in the screen, and genes from the canonical oxidative stress pathway, to modify the toxicity of Aβ was confirmed in flies by specifically over-expressing each transgene in combination with wild-type or Arctic1–42. In this way we assessed candidate genes from the GS element screen, i.e. the heavy and light chains of Drosophila ferritin (Fer1HC and Fer2LC) and carbonyl reductase by over-expression of the Drosophila enzyme Sniffer (SNI). We also tested candidate genes including cytoplasmic CuZn-SOD1, the mitochondrial Mn-SOD2 (mitSOD2) and CAT, and GST (Fig. 4). This approach allowed comparison of the efficacy of the upstream enzymes that modulate the generation of free radicals (SOD1, mitSOD2, CAT, Fer1HC and Fer2LC) with the downstream enzymes that repair oxidative damage (GST and carbonyl reductase/SNI).

Fig. 4
Co-expression of antioxidative stress genes with Aβ increased the median survival of flies. Co-expression of both ferritin heavy (elav-Gal4 UAS-Fer1HC) and light (elav-Gal4 UAS-Fer2LC) chains prolonged the lifespan of Arctic1–42 ...

Over-expression of ferritin heavy chain resulted in a 105% increase in median survival of Arctic1–42 flies (P < 0.0001), whereas over-expression of ferritin light chain gave a 49% increase in median survival (P < 0.0001) (Fig. 4a and b). The most powerful canonical oxidative stress-related protein was CAT, which increased the median survival of Arctic1–42 flies by 56% (P < 0.0001, Fig. 4a and c). MitSOD2 prolonged median survival by 18% (P < 0.0001, Fig. 4a and d). In contrast, and contrary to our expectations, the over-expression of cytoplasmic SOD1 enhanced the toxicity of Arctic1–42. Flies expressing mutants of SOD1 that are dominant-negative for activity were then assessed to see if they had a similar modifying activity. Co-expression of both the SOD1n108 mutant and RNAi for SOD1 resulted in a 43% and 32% increase in median survival, respectively (Fig. 4a and c), indicating that it is the catalytic activity of SOD1 that potentiates the toxic effect of the Aβ. The protection afforded by CAT combined with the toxicity of SOD1 suggests that the uncompensated production of H2O2 is a vital step in the oxidative stress caused by the Aβ. An additional stress resulting from increased SOD1 activity may be the co-production of O2 by the dismutation of superoxide radicals. This oxygen may go on to generate further H2O2 by reacting with iron or copper ions that are complexed with Aβ (Huang et al., 1999a,b;).

Much smaller protective activities were observed when enzymes, specifically Drosophila carbonyl reductase (SNI) and GST, that are involved in steps further downstream in the pathway were upregulated (Fig. 4a). Control experiments, in which the antioxidative stress genes were over-expressed in flies that were identical except that they lacked the Aβ transgene, did not reveal non-specific prolongation of life (Fig. 4d). In flies expressing wild-type Aβ1–42 we were able to confirm the prolongation of lifespan by the heavy and light chains of ferritin and the toxic effect of SOD1 (data not shown).

Transgenic over-expression of single antioxidative stress genes rescues the β-amyloid peptide-induced locomotor phenotype

The modifiers were then assessed for their ability to protect against the Aβ1–42-mediated decline in locomotor function. Flies co-expressing either ferritin subunit with Arctic1–42 were significantly more mobile than flies expressing Arctic1–42 alone (Fig. 5a, elav-Gal4 UAS-Arctic Aβ1–42) from day 10 onwards, and both the heavy and light chain flies performed like control flies (Fig. 5a, elav-Gal4) up to day 25. The effect of the heavy chain was more potent, maintaining wild-type locomotor performance in Arctic1–42 flies to day 35. The behavioural effects of over-expressing the canonical antioxidative genes were in accord with their longevity data; CAT (Fig. 5b, elav-Gal4 UAS-Arctic Aβ1–42UAS-CAT) and the mitSOD2 (Fig. 5b, elav-Gal4 UAS-Arctic Aβ1–42UAS-mitSOD2) were suppressors of the Arctic1–42 locomotor phenotype, whereas the cytoplasmic SOD1 (Fig. 5b, elav-Gal4 UAS-Arctic Aβ1–42UAS-SOD1) accelerated the decline in locomotor function. Over-expression of the heavy chain of ferritin (Fig. 5c, elav-Gal4 UAS-Aβ1–42UAS-FerHC) and CAT (Fig. 5c, elav-Gal4 UAS-Aβ1–42UAS-CAT) had similar beneficial effects in flies expressing wild-type Aβ1–42 (Fig. 5c, elav-Gal4 UAS-Aβ1–42). As expected, flies co-expressing SOD1 (Fig. 5c, elav-Gal4 UAS-Aβ1–42UAS-SOD1) with wild-type Aβ1–42 demonstrated an accelerated decline in locomotor function. The co-expression of GST (Fig. 5d, elav-Gal4 UAS-Arctic Aβ1–42UAS-GST) gave a weak but significant improvement in locomotor function from day 10 onwards. Carbonyl reductase (Fig. 5d, elav-Gal4 UAS-Arctic Aβ1–42UAS-SNI) did not significantly improve locomotor function.

Fig. 5
Over-expressing oxidative stress-related genes modified the Aβ-induced locomotor phenotype. Flies expressing Arctic1–42 (a, UAS-Arctic Aβ1–42) exhibited a progressive decline in locomotor function becoming immobile ...

The metal chelator clioquinol reduces β-amyloid peptide-mediated neuronal toxicity and specifically reduces iron levels in the brain

That the protection against Aβ toxicity afforded by ferritin is mediated in part by the chelation of Fe2+ and Fe3+ ions was investigated by treating flies expressing Aβ1–40, wild-type Aβ1–42 and Arctic1–42 with the metal chelator clioquinol (Kaur et al., 2003) (Fig. 6). Although clioquinol had no effect on the longevity of flies expressing the non-toxic Aβ1–40 (Fig. 6a, circles) there was, in contrast, a clear dose-related increase in longevity for Arctic1–42 (Fig. 6a, diamonds) flies. Expression of wild-type Aβ1–42 (Fig. 6a, triangles) gave an intermediate response to clioquinol treatment with an optimum dose of approximately of 20 μm. The degree of functional rescue in flies expressing Arctic1–42 closely reflected the concentration of iron in the extracts of fly heads (Fig. 6b). We found that the presence of Arctic1–42 (Fig. 6b, triangles) increased the uptake of iron as compared with control flies (Fig. 6b, circles), a striking effect that is only completely reversed by treatment with 200 μm clioquinol. The levels of zinc (1.5–1.7 mm) were the same in Arctic1–42-expressing and control flies and remained unchanged following clioquinol treatment. Copper could not be detected in the head extracts.

Fig. 6
The iron-chelating compound clioquinol was added to the fly food at final concentrations of 2, 20 and 200 μm and its effects on the Aβ-associated longevity phenotype and the accumulation of iron in the fly brain was assessed. The median ...

Oxidative damage in flies expressing β-amyloid peptide is reduced by ferritin and increased by superoxide dismutase 1

The quantity of carbonyl groups was assessed in flies expressing Arctic1–42 in the presence or absence of the heavy and light chains of ferritin. The expression of Arctic1–42 significantly increased the concentration of carbonyl groups (Fig. 7a, elav-Gal4 w1118 vs. elav-Gal4 UAS-Arctic Aβ1–42, P < 0.001) but this was reduced by 30% following the co-expression of ferritin light chain (Fig. 7a, elav-Gal4 UAS-Arctic Aβ1–42UAS-FerLC and elav-Gal4 UAS-Arctic Aβ1–42, P < 0.01). Remarkably, ferritin heavy chain (Fig. 7a, elav-Gal4 UAS-Arctic Aβ1–42UAS-FerHC) reduced the carbonyl level (P < 0.001) almost to that of control flies (Fig. 7a, control elav-Gal4 UAS-Arctic Aβ1–42). These findings are consistent with ferritin exerting its suppression of the Aβ phenotype by an antioxidant effect. Conversely, the enhancer effect of SOD1 was accompanied by a significant increase in carbonyl levels in Arctic1–42 flies (Fig. 7a, elav-Gal4 UAS-Arctic Aβ1–42 vs. elav-Gal4 UAS-Arctic Aβ1–42UAS-SOD1, P < 0.01). These data suggest that the rapid production of H2O2 by cytoplasmic SOD1 can overwhelm endogenous CAT resulting in the Fe2+-mediated generation of the hydroxyl radical (Fenton reaction) that damages proteins as demonstrated by the increased carbonyl load.

Fig. 7
Oxidative stress mediated by the Arctic1–42 is reversed by ferritin despite an increase in the levels of Aβ1–42. A reduction in carbonyl load was apparent with co-expression of the light chain of ferritin (a, UAS-FerLC ...

Ferritin subunits suppress the toxicity of β-amyloid peptide1–42 despite increased levels of β-amyloid peptide1–42 in the brain

The effect of over-expressing antioxidant transgenes on the level of Aβ1–42 in the brains of flies was assessed by an enzyme-linked immunosorbent assay that measured 5 m guanidinium hydrochloride-soluble Aβ1–42. Six independent lines of flies expressing Arctic1–42 and carrying GS elements that cause over-expression of ferritin subunits were shown to accumulate significantly higher levels of Aβ1–42 in their brains (65 pm in control flies vs. 100 pm for GS elements co-expressing the light chain gene, P < 0.01, and 141 pm over-expressing the heavy chain gene, P < 0.01). This elevation of Aβ1–42 when over-expressing ferritin subunits was confirmed in flies co-expressing Aβ1–42 with UAS-linked transgenes for either the heavy or light chains (Fig. 7b). In contrast, the levels of Aβ1–42 were unaffected by the co-expression of CAT or the over-expression, or the knockdown, of SOD-1. Thus, the rescue mediated by over-expression of oxidative stress genes was not mediated by suppression of the Aβ1–42.

Discussion

Two genome-wide genetic screens were used in our Drosophila model of AD to identify genes that play an important role in the toxicity of the Aβ. The first screen, using Drosophila cDNA microarrays, quantified the changes in gene transcription that occurred in response to the expression of Aβ1–42 as compared with Aβ1–40. The second complementary screen, using a library of flies with unique GS element insertions, identified genes, which when transcriptionally up- or downregulated modified the lifespan of flies expressing Aβ1–42. Taken as a whole the GS screen was remarkable for the number of genes adjacent to modifying P-elements that had redox or antioxidant activities. Similarly, the cDNA microarray studies showed that redox-associated transcripts were the most robustly represented functional group of genes that were differentially regulated by Aβ1–42 expression. This combination of observations is powerful because, whereas the microarray data provide genetic evidence of a response by the brain to oxidative stress, the GS screen, in contrast, points to a direct pathogenic role for oxidative stress in the generation of Aβ-related phenotypes. The role of oxidation in causing disease was further emphasized by the remarkably close correlation between the severity of the phenotypes observed for Aβ1–40, Aβ1–42 and Arctic1–42 and the oxidative modification of brain proteins as determined by carbonyl levels. These findings are concordant with the raised levels of oxidized proteins and lipids in post-mortem AD brains where it is known that oxidative damage is present from the earliest clinical stages of the disease (Nunomura et al., 2001; Markesbery et al., 2005). Our work therefore represents an advance on current clinical work and mouse models that have not clarified whether oxidative stress plays a direct role in the pathogenesis of AD or whether it is a consequence of the disease process. Indeed, the view that oxidative stress is a bystander effect is supported by clinical trial data that show, despite early encouraging data from observational studies (Morris et al., 2002a,b; Zandi et al., 2004) and one prospective trial (Sano et al., 1997), that the antioxidant vitamin E is not able to prevent the onset of dementia or prevent progression of established disease (Petersen et al., 2005).

In this work we have undertaken a molecular dissection of the oxidative stress pathway (Fig. 8) in order to gain a more detailed understanding of which oxidative species were of particular importance in Aβ-mediated neurodegeneration. This work has emphasized three main sources of oxidative stress: firstly, the generation of hydroxyl radicals via the Fenton reaction (Smith et al., 1997; Dikalov et al., 2004), secondly, mitochondrial superoxide levels and thirdly, reactive aldehyde species produced by lipid oxidation. Although each of these sources contributes significantly to oxidative stress, the conversion of H2O2 to the hydroxyl radical is the most important toxic process. The most immediate support for this comes from the profound protection afforded to flies when CAT is co-expressed with Aβ1–42 because hydroxyl radical generation will predictably be abolished by converting H2O2 to water.

Fig. 8
Model of Aβ-mediated oxidative stress. CuZn-SOD1 breaks down superoxide free radicals (O2−) in the cytoplasm to produce H2O2 and molecular oxygen (O2). In the presence of oxygen, Fe2+ cycles back to Fe3+ by the Aβ to produce H ...

Only co-expression of ferritin subunits offered a more effective rescue of the longevity phenotype than CAT. Ferritin proteins are highly conserved in evolution and have been extensively characterized (Harrison & Arosio, 1996). Functional ferritin complexes may be composed of various proportions of heavy and light subunits. For instance, in the human, ferritin from the brain is predominantly composed of heavy chain, whereas in the liver the ferritin has a high proportion of light chain. In our model, the over-expression of Fer1HC or Fer2LC occurred in the context of endogenous Fer1HC and Fer2LC expression. Thus, in our transgenic animals we should produce ferritin complexes composed of both subunits but with one or the other representing the major component. Remarkably, ferritin heavy chain was able to restore the longevity and locomotor phenotype of flies expressing the highly toxic Arctic1–42 to that of control flies. That this rescue was mediated by an antioxidant effect is supported by the reductions in carbonyl levels in the brains of flies co-expressing either the light or, again more potently, heavy chain of ferritin. It is likely that the sequestration of iron by both ferritin subunits, and the conversion of Fe2+ to Fe3+ by the heavy chain, slows hydroxyl radical production. Our data show for the first time that it is likely to be the removal of Fe2+, by the ferroxidase activity of the heavy chain, that is specifically beneficial. Previous in-vitro data have shown that synthetic Aβ can directly generate H2O2 in the presence of metal ions (Huang et al., 1999a; Tabner et al., 2005) and in particular iron (Khan et al., 2006). Using molecular oxygen as a substrate, the production of H2O2 by synthetic Aβ depends on Fe2+ ions generated via a redox cycling of iron (Huang et al., 1999a; Khan et al., 2006). Thus, it is possible that ferritin can not only prevent the Fenton reaction but additionally the Fe2+-scavenging activity of ferritin heavy chain can also protect neurones against the intrinsic redox properties of Aβ (Fig. 8).

The powerful antioxidant properties of ferritin can also rescue the locomotor deficits associated with Aβ expression despite an accompanying twofold increase in Aβ load. These data make the modulation of brain iron metabolism an attractive therapeutic target not least because the concentration of Fe2+ in amyloid plaques is 1 mm, almost three times the normal level (Bush, 2003). Furthermore, clioquinol, a metal chelator, reduces plaque deposition in mouse models of AD (Cherny et al., 2001), is safe in clinical trials (Ritchie et al., 2003) and, as we show here, specifically reduces iron levels in the brain and prolongs the survival of flies expressing Arctic Aβ1–42.

Oxidative stress has been linked with mitochondrial dysfunction in several neurodegenerative disorders including AD and also Parkinson’s and Huntington’s diseases (Bowling & Beal, 1995; Maier & Chan, 2002). In AD there is evidence that soluble oligomeric aggregates of Aβ may damage the insulating properties of the neuronal plasma membrane (Kayed et al., 2004) resulting in calcium influx (Demuro et al., 2005). Mitochondria from patients with AD are impaired in their ability to buffer calcium influxes and also show defects in their respiratory complexes, particularly complex IV, resulting in increased generation of reactive oxygen species (Sheehan et al., 1997; Abramov et al., 2004). Our data are consistent with these hypotheses showing that over-expression of the mitochondrial superoxide scavenging enzyme mitSOD2 offered modest but significant protection against Aβ toxicity.

It was surprising that the co-expression of SOD1 enhanced the toxicity of Aβ1–42. This result underlines the low toxicity of the superoxide anion and again emphasizes the importance of H2O2 in the oxidative stress pathway. By showing that dominant negative mutants of SOD1 prolong the life of the AD flies, our findings demonstrate that SOD1 catalytic activity mediates its toxicity. Furthermore, RNAi knockdown of SOD1 protein expression did not provide any additional rescue, indicating that our data are not confounded by toxicity caused by SOD1 misfolding and aggregation as is proposed for SOD1-linked familial amyotrophic lateral sclerosis (Lynch et al., 2004). The finding of elevated carbonyl levels in the brains of flies co-expressing SOD1 and Arctic1–42 suggests that rapid dismutation of superoxide can overwhelm the capacity of endogenous CAT to remove the consequent H2O2. In addition, the dismutation of superoxide radicals releases molecular oxygen that may become a substrate for the Aβ-dependent production of H2O2 (Fig. 8). Nevertheless, the enhancer activity of SOD1 in Drosophila was unexpected because previous studies in a mouse model of AD showed that SOD1 knockdown caused increased activation of apoptotic pathways (Chen et al., 2005). Further work is required to determine whether SOD1 upregulation is toxic in a mammalian model of AD.

Finally, we have evaluated the effect of two enzymes that are involved in the detoxification of reactive aldehydes (GST and carbonyl reductase). Reactive aldehydes, such as 4-oxonon-2-enal, result from hydroxyl radical-mediated lipid oxidation and accumulate in the brains of patients with AD (Lovell et al., 1997; Markesbery & Lovell, 1998). Despite being less reactive than the hydroxyl radical, the greater stability of the aldehydes permits damage to proteins and DNA over longer times and distances within the cell. We have shown that over-expression of GST or carbonyl reductase gave only a modest rescue of Aβ toxicity, suggesting that lipid damage is indeed downstream of the most important toxic events.

In summary, we have used genetic screens and a candidate gene approach to dissect the contribution of reactive oxygen species to the toxicity of Aβ in our Drosophila model of AD. We have found that the primary oxidative stressors are likely to be H2O2 and the consequent hydroxyl radical. Preventing oxidative stress, specifically by manipulating iron metabolism, provides a powerful strategy for reducing Aβ toxicity in AD.

Acknowledgments

This work was supported by the Wellcome Trust, Medical Research Council (UK), Papworth NHS Trust and an industrial collaboration grant between Merck Sharp & Dohme Ltd and the University of Cambridge. R.M.P. was funded by a studentship as part of an educational agreement between Merck Sharp & Dohme Ltd and the University of Cambridge. We are grateful to Professor M. Ashburner, Dr S. Russell and the Department of Genetics, University of Cambridge for the use of the fly laboratory facilities and thank Dr S. Imarisio (Cambridge Institute for Medical Research) for her comments on the manuscript.

Glossary

Abbreviations

β-amyloid peptide
AD
Alzheimer’s disease
CAT
catalase
Fer1HC
ferritin 1 heavy chain
Fer2LC
ferritin 2 light chain
GS
Gene Search
GST
glutathione-S-transferase
mitSOD2
mitochondrial Mn-superoxide dismutase 2
SNI
Sniffer
SOD
superoxide dismutase
UAS
upstream activating sequence

Supporting Information

Additional supporting information may be found in the online version of this article:

Fig. S1. (a) Experimental design for the Affymetrix chip analysis of differential gene transcription.

Fig. S2. In control experiments, the expression of the key modifiers of A toxicity in the absence of A did not have a beneficial effect on locomotor function at any time point.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

References

  • Abramov AY, Canevari L, Duchen MR. Beta-amyloid peptides induce mitochondrial dysfunction and oxidative stress in astrocytes and death of neurons through activation of NADPH oxidase. J. Neurosci. 2004;24:565–575. [PubMed]
  • Alamdari DH, Kostidou E, Paletas K, Sarigianni M, Konstas AG, Karapiperidou A, Koliakos G. High sensitivity enzyme-linked immunosorbent assay (ELISA) method for measuring protein carbonyl in samples with low amounts of protein. Free Radic. Biol. Med. 2005;39:1362–1367. [PubMed]
  • Anderson PR, Kirby K, Hilliker AJ, Phillips JP. RNAi-mediated suppression of the mitochondrial iron chaperone, frataxin, in Drosophila. Hum. Mol. Genet. 2005;14:3397–3405. [PubMed]
  • Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 2000;25:25–29. [PMC free article] [PubMed]
  • Botella JA, Ulschmid JK, Gruenewald C, Moehle C, Kretzschmar D, Becker K, Schneuwly S. The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration. Curr. Biol. 2004;14:782–786. [PubMed]
  • Bowling AC, Beal MF. Bioenergetic and oxidative stress in neurodegenerative diseases. Life Sci. 1995;56:1151–1171. [PubMed]
  • Bush AI. The metallobiology of Alzheimer’s disease. Trends Neurosci. 2003;26:207–214. [PubMed]
  • Cao W, Song HJ, Gangi T, Kelkar A, Antani I, Garza D, Konsolaki M. Identification of novel genes that modify phenotypes induced by Alzheimer’s β-amyloid overexpression in Drosophila. Genetics. 2008;178:1457–1471. [PMC free article] [PubMed]
  • Chen Z, Duan RS, Lepecheur M, Paly E, London J, Zhu J. SOD-1 inhibits FAS expression in cortex of APP transgenic mice. Apoptosis. 2005;10:499–502. [PubMed]
  • Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA, Barnham KJ, Volitakis I, Fraser FW, Kim Y, Huang X, Goldstein LE, Moir RD, Lim JT, Beyreuther K, Zheng H, Tanzi RE, Masters CL, Bush AI. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer’s disease transgenic mice. Neuron. 2001;30:665–676. [PubMed]
  • Crowther DC, Kinghorn KJ, Miranda E, Page R, Curry JA, Duthie FA, Gubb DC, Lomas DA. Intraneuronal Aβ, non-amyloid aggregates and neurodegeneration in a Drosophila model of Alzheimer’s disease. Neuroscience. 2005;132:123–135. [PubMed]
  • Demuro A, Mina E, Kayed R, Milton SC, Parker I, Glabe CG. Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers. J. Biol. Chem. 2005;280:17294–17300. [PubMed]
  • Dikalov SI, Vitek MP, Mason RP. Cupric-amyloid beta peptide complex stimulates oxidation of ascorbate and generation of hydroxyl radical. Free Radic. Biol. Med. 2004;36:340–347. [PubMed]
  • Harrison PM, Arosio P. The ferritins: molecular properties, iron storage function and cellular regulation. Biochim. Biophys. Acta. 1996;1275:161–203. [PubMed]
  • Huang X, Atwood CS, Hartshorn MA, Multhaup G, Goldstein LE, Scarpa RC, Cuajungco MP, Gray DN, Lim J, Moir RD, Tanzi RE, Bush AI. The A beta peptide of Alzheimer’s disease directly produces hydrogen peroxide through metal ion reduction. Biochemistry. 1999a;38:7609–7616. [PubMed]
  • Huang X, Cuajungco MP, Atwood CS, Hartshorn MA, Tyndall JD, Hanson GR, Stokes KC, Leopold M, Multhaup G, Goldstein LE, Scarpa RC, Saunders AJ, Lim J, Moir RD, Glabe C, Bowden EF, Masters CL, Fairlie DP, Tanzi RE, Bush AI. Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction. J. Biol. Chem. 1999b;274:37111–37116. [PubMed]
  • Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 2003;31:e15. [PMC free article] [PubMed]
  • Jo DG, Lee JY, Hong YM, Song S, Mook-Jung I, Koh JY, Jung YK. Induction of pro-apoptotic calsenilin/DREAM/KChIP3 in Alzheimer’s disease and cultured neurons after amyloid-beta exposure. J. Neurochem. 2004;88:1570. [PubMed]
  • Kaur D, Yantiri F, Rajagopalan S, Kumar J, Mo JQ, Boonplueang R, Viswanath V, Jacobs R, Yang L, Beal MF, DiMonte D, Volitaskis I, Ellerby L, Cherny RA, Bush AI, Andersen JK. Genetic or pharmacological iron chelation prevents MPTP-induced neurotoxicity in vivo: a novel therapy for Parkinson’s disease. Neuron. 2003;37:899–909. [PubMed]
  • Kayed R, Sokolov Y, Edmonds B, McIntire TM, Milton SC, Hall JE, Glabe CG. Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfolding diseases. J. Biol. Chem. 2004;279:46363–46366. [PubMed]
  • Khan A, Dobson JP, Exley C. Redox cycling of iron by Abeta42. Free Radic. Biol. Med. 2006;40:557–569. [PubMed]
  • Kienlen-Campard P, Miolet S, Tasiaux B, Octave JN. Intracellular amyloid-beta 1–42, but not extracellular soluble amyloid-beta peptides, induces neuronal apoptosis. J. Biol. Chem. 2002;277:15666–15670. [PubMed]
  • Koo EH, Squazzo SL. Evidence that production and release of amyloid beta-protein involves the endocytic pathway. J. Biol. Chem. 1994;269:17386–17389. [PubMed]
  • Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Liosatos M, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch CE, Krafft GA, Klein WL. Diffusible, nonfibrillar ligands derived from Abeta1–42 are potent central nervous system neurotoxins. Proc. Natl Acad. Sci. USA. 1998;95:6448–6453. [PMC free article] [PubMed]
  • Lashuel HA, Hartley D, Petre BM, Walz T, Lansbury PT., Jr Neurodegenerative disease: amyloid pores from pathogenic mutations. Nature. 2002;418:291. [PubMed]
  • Lovell MA, Ehmann WD, Mattson MP, Markesbery WR. Elevated 4-hydroxynonenal in ventricular fluid in Alzheimer’s disease. Neurobiol. Aging. 1997;18:457–461. [PubMed]
  • Luheshi LM, Tartaglia GG, Brorsson AC, Pawar AP, Watson IE, Chiti F, Vendruscolo M, Lomas DA, Dobson CM, Crowther DC. Systematic in vivo analysis of the intrinsic determinants of amyloid beta pathogenicity. PLoS Biol. 2007;5:e290. [PMC free article] [PubMed]
  • Lynch SM, Boswell SA, Colon W. Kinetic stability of Cu/Zn superoxide dismutase is dependent on its metal ligands: implications for ALS. Biochemistry. 2004;43:16525–16531. [PubMed]
  • Maier CM, Chan PH. Role of superoxide dismutases in oxidative damage and neurodegenerative disorders. Neuroscientist. 2002;8:323–334. [PubMed]
  • Markesbery WR, Lovell MA. Four-hydroxynonenal, a product of lipid peroxidation, is increased in the brain in Alzheimer’s disease. Neurobiol. Aging. 1998;19:33–36. [PubMed]
  • Markesbery WR, Kryscio RJ, Lovell MA, Morrow JD. Lipid peroxidation is an early event in the brain in amnestic mild cognitive impairment. Ann. Neurol. 2005;58:730–735. [PubMed]
  • Missirlis F, Hu J, Kirby K, Hilliker AJ, Rouault TA, Phillips JP. Compartment-specific protection of iron-sulfur proteins by superoxide dismutase. J. Biol. Chem. 2003;278:47365–47369. [PubMed]
  • Morris MC, Evans DA, Bienias JL, Tangney CC, Bennett DA, Aggarwal N, Wilson RS, Scherr PA. Dietary intake of antioxidant nutrients and the risk of incident Alzheimer disease in a biracial community study. JAMA. 2002a;287:3230–3237. [PubMed]
  • Morris MC, Evans DA, Bienias JL, Tangney CC, Wilson RS. Vitamin E and cognitive decline in older persons. Arch. Neurol. 2002b;59:1125–1132. [PubMed]
  • Nilsberth C, Westlind-Danielsson A, Eckman CB, Condron MM, Axelman K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG, Naslund J, Lannfelt L. The ‘Arctic’ APP mutation (E693G) causes Alzheimer’s disease by enhanced Abeta protofibril formation. Nat. Neurosci. 2001;4:887–893. [PubMed]
  • Nunomura A, Perry G, Aliev G, Hirai K, Takeda A, Balraj EK, Jones PK, Ghanbari H, Wataya T, Shimohama S, Chiba S, Atwood CS, Petersen RB, Smith MA. Oxidative damage is the earliest event in Alzheimer disease. J. Neuropathol. Exp. Neurol. 2001;60:759–767. [PubMed]
  • Petersen RC, Thomas RG, Grundman M, Bennett D, Doody R, Ferris S, Galasko D, Jin S, Kaye J, Levey A, Pfeiffer E, Sano M, van Dyck CH, Thal LJ. Vitamin E and donepezil for the treatment of mild cognitive impairment. N. Engl. J. Med. 2005;352:2379–2388. [PubMed]
  • Phillips JP, Tainer JA, Getzoff ED, Boulianne GL, Kirby K, Hilliker AJ. Subunit-destabilizing mutations in Drosophila copper/zinc superoxide dismutase: neuropathology and a model of dimer dysequilibrium. Proc. Natl Acad. Sci. USA. 1995;92:8574–8578. [PMC free article] [PubMed]
  • Refolo LM, Sambamurti K, Efthimiopoulos S, Pappolla MA, Robakis NK. Evidence that secretase cleavage of cell surface Alzheimer amyloid precursor occurs after normal endocytic internalization. J. Neurosci. Res. 1995;40:694–706. [PubMed]
  • Ritchie CW, Bush AI, Mackinnon A, Macfarlane S, Mastwyk M, MacGregor L, Kiers L, Cherny R, Li QX, Tammer A, Carrington D, Mavros C, Volitakis I, Xilinas M, Ames D, Davis S, Beyreuther K, Tanzi RE, Masters CL. Metal-protein attenuation with iodochlorhydroxyquin (clioquinol) targeting Abeta amyloid deposition and toxicity in Alzheimer disease: a pilot phase 2 clinical trial. Arch. Neurol. 2003;60:1685–1691. [PubMed]
  • Rival T, Soustelle L, Strambi C, Besson MT, Iche M, Birman S. Decreasing glutamate buffering capacity triggers oxidative stress and neuropil degeneration in the Drosophila brain. Curr. Biol. 2004;14:599–605. [PubMed]
  • Sano M, Ernesto C, Thomas RG, Klauber MR, Schafer K, Grundman M, Woodbury P, Growdon J, Cotman CW, Pfeiffer E, Schneider LS, Thal LJ. A controlled trial of selegiline, alpha-tocopherol, or both as treatment for Alzheimer’s disease. The Alzheimer’s Disease Cooperative Study. N. Engl. J. Med. 1997;336:1216–1222. [PubMed]
  • Sheehan JP, Swerdlow RH, Miller SW, Davis RE, Parks JK, Parker WD, Tuttle JB. Calcium homeostasis and reactive oxygen species production in cells transformed by mitochondria from individuals with sporadic Alzheimer’s disease. J. Neurosci. 1997;17:4612–4622. [PubMed]
  • Smith MA, Harris PL, Sayre LM, Perry G. Iron accumulation in Alzheimer disease is a source of redox-generated free radicals. Proc. Natl Acad. Sci. USA. 1997;94:9866–9868. [PMC free article] [PubMed]
  • Tabner BJ, El-Agnaf OM, Turnbull S, German MJ, Paleologou KE, Hayashi Y, Cooper LJ, Fullwood NJ, Allsop D. Hydrogen peroxide is generated during the very early stages of aggregation of the amyloid peptides implicated in Alzheimer Disease and familial British dementia. J. Biol. Chem. 2005;280:35789–35792. [PubMed]
  • Toba G, Ohsako T, Miyata N, Ohtsuka T, Seong KH, Aigaki T. The gene search system. A method for efficient detection and rapid molecular identification of genes in Drosophila melanogaster. Genetics. 1999;151:725–737. [PMC free article] [PubMed]
  • Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, Rowan MJ, Selkoe DJ. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature. 2002;416:535–539. [PubMed]
  • Wei W, Norton DD, Wang X, Kusiak JW. Abeta 17-42 in Alzheimer’s disease activates JNK and caspase-8 leading to neuronal apoptosis. Brain. 2002;125:2036–2043. [PubMed]
  • Whitworth AJ, Theodore DA, Greene JC, Benes H, Wes PD, Pallanck LJ. Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson’s disease. Proc. Natl Acad. Sci. USA. 2005;102:8024–8029. [PMC free article] [PubMed]
  • Zandi PP, Anthony JC, Khachaturian AS, Stone SV, Gustafson D, Tschanz JT, Norton MC, Welsh-Bohmer KA, Breitner JC. Reduced risk of Alzheimer disease in users of antioxidant vitamin supplements: the Cache County Study. Arch. Neurol. 2004;61:82–88. [PubMed]
  • Zhang B, Schmoyer D, Kirov S, Snoddy J. GOTree Machine (GOTM): a web-based platform for interpreting sets of interesting genes using Gene Ontology hierarchies. BMC Bioinformatics. 2004;5:16. [PMC free article] [PubMed]

Articles from Wiley-Blackwell Online Open are provided here courtesy of Wiley-Blackwell, John Wiley & Sons

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...