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Redox-active iron mediates amyloid-β toxicity

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Abstract

While amyloid-β toxicity is mediated by oxidative stress and can be attenuated by antioxidants, the actual biochemical mechanism underlying neurotoxicity remains to be established. However, since aggregated amyloid-β can interact with transition metals, such as iron, both in vitro and in vivo, we suspected that bound iron might be the mediator of toxicity such that holo- and apo-amyloid would have differential effects on cellular viability. Here we demonstrate that when amyloid-β is pretreated with the iron chelator deferoxamine, neuronal toxicity is significantly attenuated while conversely, incubation of holo-amyloid-β with excess free iron restores toxicity to original levels. These data, taken together with the known sequelae of amyloid-β, suggest that the toxicity of amyloid-β is mediated, at least in part, via redox-active iron that precipitates lipid peroxidation and cellular oxidative stress.

Introduction

Alzheimer disease (AD), the leading cause of dementia in the elderly, is a disorder that has been largely defined on a pathological basis. For this reason, one of the major focuses of AD-related basic and clinical research has centered on the neurotoxicity of the amyloid-β peptide, whose burden in the brain is correlated with dementia [1], [2], and which is a dominant criteria for diagnosis. An overwhelming number of studies have pointed to the fact that amyloid-β is inherently toxic to neurons and clonal cell lines in culture [3], [4], [5]. Toxicity to cells, together with evidence that amyloid-β deposits are found in regions of the brain susceptible to the neurodegenerative processes of AD and that amyloid-β production is increased in all of the inherited forms of AD, has led to the widely held belief that amyloid-β is a key mediator in the pathogenesis of AD [6].

Numerous studies support the idea that an oxidative event is critical for amyloid-β neurotoxicity (reviewed in [5]) and, in this regard, the cytotoxic effects of amyloid-β can be attenuated by application of antioxidants and free-radical scavengers such as vitamin E [7]. The prooxidant potential of amyloid-β is also supported by in vivo evidence where deposits are associated with oxidative damage [8], [9] and such damage is, like amyloid-β deposition [6], viewed as an extremely proximal event in disease pathogenesis [10], [11]. However, while it is clear that amyloid-β causes oxidative stress and that toxicity can be attenuated by antioxidants, the precise mechanism by which amyloid-β leads to increased oxidative stress has remained elusive. Indeed, while studies have suggested that the neurotoxicity of aggregated amyloid-β is mediated by its ability to induce oxidative stress via the spontaneous generation of free radicals and reactive oxygen species [12], this proposition has been questioned on theoretical and methodological grounds [13], [14]. We hypothesized that the prooxidant effects of amyloid-β were likely to be mediated by its interaction with redox-active metals such as iron or copper. In this vein, both iron and copper have an unusually high affinity for amyloid-β [15], [16] and amyloid-β has been shown to be capable of reducing these metal ions with the subsequent production of hydrogen peroxide and oxidized amyloid-β [17], [18], [19]. The physiological relevance of transition metals to amyloid-β is demonstrated by their concurrent accumulation in amyloid-β deposits in AD [20], [21] as well as in transgenic mouse models [9].

In this study, we specifically address whether amyloid-β toxicity is mediated via its direct interaction with redox-active metals.

Section snippets

Cell culture

The human neuroblastoma cell line M17 was maintained at 37°C/5.0% CO2 in serum-free Opti-MEM media (Life Technologies, Gaithersburg, MD, USA) supplemented with 5% donor calf serum and 1% penicillin/streptomycin with fungizone (Life Technologies). Cells were plated in 96 well microtiter plates at a density of 1 × 10−4 cells/well approximately 18 h before the addition of amyloid-β peptide. The cultures were then treated with various concentrations (100 nM–10 μM) of (i) amyloid-β1–42; (ii)

Results and discussions

To evaluate the role of iron in amyloid-β-mediated neurotoxicity we pretreated synthetic amyloid-β fibrils with the iron chelator, deferoxamine, and removed the chelated iron by washing the fibril pellet prior to cell culture toxicity studies. Interestingly, we found that pretreatment with deferoxamine significantly decreases the in vitro neurotoxicity of pre-aggregated amyloid-β1–42 (Fig. 1). Indicative of a key role for iron removal in this attenuated toxicity, incubation of

Acknowledgements

This work was supported through a grant from the National Institutes of Health (NS38648).

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