There is clear evidence implicating oxidative stress in the pathology of many different neurodegenerative diseases. ROS (reactive oxygen species) are the primary mediators of oxidative stress and many of the aggregating proteins and peptides associated with neurodegenerative disease can generate hydrogen peroxide, a key ROS, apparently through interactions with redox-active metal ions. Our recent results suggest that ROS are generated during the very early stages of protein aggregation, when protofibrils or soluble oligomers are present, but in the absence of mature amyloid fibrils. The generation of ROS during early-stage protein aggregation may be a common, fundamental molecular mechanism underlying the pathogenesis of oxidative damage, neurodegeneration and cell death in several different neurodegenerative diseases. Drugs that specifically target this process could be useful in the future therapy of these diseases.
The formation of extracellular deposits of ‘amyloid’ fibrils and/or intracellular inclusions containing protein fibrils is one of the key pathological features of many different human neurodegenerative diseases, including AD (Alzheimer's disease), PD (Parkinson's disease), the ‘prion’ dementias (or transmissible spongiform encephalopathies), the ‘tauopathies’, Huntington's disease and other trinucleotide repeat disorders, motor neuron disease, some rare genetic forms of neurodegenerative disease (e.g. the familial British and Danish dementias) and other related brain disorders. Convergent biochemical and genetic evidence points to the likely seminal importance of these protein deposits in disease pathogenesis (see , for a review). Disease-causing mutations have been found in the genes encoding ‘all’ of the aggregating proteins associated with these neurodegenerative diseases [Aβ (β-amyloid), α-synuclein, PrP (prion protein), tau, huntingtin, ABri (British dementia peptide) and ADan (Danish dementia peptide), Cu/Zn superoxide dismutase]. The resulting mutant proteins often show an increased propensity to form aggregates, which are toxic to cells in culture (see below), suggesting a possible direct link between the formation of protein deposits in the brain and degeneration of the nervous system. Because protein aggregation is concentration-dependent, the expression of abnormally high levels of a particular wild-type protein, due to gene duplication or triplication, can also cause neurodegenerative disease. Transgenic mice expressing individual wild-type or mutant aggregating proteins (or, as in many recent studies, a combination of more than one protein) can exhibit most, if not all, of the important histopathological, neurochemical and behavioural features of the relevant human disease [2–7]. However, some transgenic mouse models of AD develop considerable quantities of Aβ deposits in the brain but fail to show the anticipated extent and pattern of neuronal cell loss . This has cast considerable doubt on the idea that Aβ is responsible for neuronal cell death in AD, but could be explained by the fact that the rodent brain is not as susceptible as the aging human brain to the toxic effects of Aβ . Also, the absence of neuronal cell death in these AD transgenic mice contrasts markedly with the toxic effects of Aβ on cultured neuronal cells. Numerous studies have shown that pre-aggregated Aβ is toxic to cells, when added to the culture medium at low micromolar concentrations. The precise molecular mechanisms responsible for this effect are unclear, but cell death appears to be due to calcium ion influx and the induction of oxidative free radical damage, usually followed by apopotosis. Earlier studies tended to emphasize the role of fully formed amyloid fibrils in this toxicity, but recent attention has focused on the possibility that early protein assemblies, of various types, referred to as ‘protofibrils’, ‘annular protofibrils’ (ringshaped structures), ‘soluble oligomers’, ‘Aβ-derived diffusible ligands’ or ‘globular neurotoxins’, could be responsible [9–16]. Similar types of early aggregates formed from other disease-associated proteins are also toxic to cultured cells, possibly through a common conformation-dependent mechanism [11,12]. A recent report suggests that this could involve calcium dysregulation and disruption of the plasma membrane , but the detailed molecular mechanisms responsible for this effect, and the relevance of these studies to neurodegeneration and cell death in vivo, are still not clear. Recently, it has also been reported that very low concentrations (1–2 nM) of cell-derived ‘soluble oligomers’ of Aβ can inhibit LTP (long-term potentiation)  and disrupt learned behaviour in rats , suggesting that early forms of protein aggregate could also act as potent modulators of synaptic plasticity. However, such low concentrations of soluble oligomers have not been shown to induce neuronal cell death, and so the relevance of these observations to neurodegeneration in vivo is, again, uncertain.
Protein aggregation and oxidative stress
There is mounting evidence for a major contribution played by oxidative stress in the pathology of AD and several other neurodegenerative diseases (reviewed in [20–25]). Oxidative stress is due to an imbalance between the production of ROS (reactive oxygen species) (including superoxide, hydrogen peroxide and the hydroxyl radical) and the ability of antioxidant defences to cope with ROS production. Evidence for oxidative stress in neurodegenerative diseases covers features such as increased levels of redox-active transition metal ions and the detection in the brain of products of lipid peroxidation and DNA and protein oxidation. Some researchers have stressed the fact that this type of oxidative damage could precede and even precipitate the formation of Aβ plaques in AD . However, Aβ has been reported to generate hydrogen peroxide, a key ROS, from molecular oxygen, through electron transfer interactions involving bound redox-active Cu(II) and Fe(III) . Hydrogen peroxide is readily converted into the aggressive hydroxyl radical by Fenton chemistry, and these two ROS could be responsible for the early oxidative damage seen in AD.
Generation of hydrogen peroxide
The production of hydrogen peroxide from Aβ has now been confirmed in a number of independent studies, including our own [27–30]. Some of these studies have shown that the amount of hydrogen peroxide generated by Aβ can be enhanced by co-incubation of the peptide with a reducing substrate, which becomes oxidized in the process [28–30]. If this substrate is cholesterol, the resulting oxidation product is 7β-hydroxycholesterol, which is proapoptotic and neurotoxic at nanomolar concentrations, and so could also contribute to oxidative brain damage in AD .
We have confirmed the ability of Aβ to generate hydrogen peroxide by using ESR spectroscopy in conjunction with the spin trapping technique  (Figure 1). Any hydrogen peroxide formed during peptide incubation can be converted into hydroxyl radicals, via the Fenton reaction, upon addition of Fe(II). The resulting radicals are then trapped by DMPO (5,5-dimethyl-1-pyrroline N-oxide) to form the DMPO-OH (DMPO hydroxyl radical adduct), which has a uniquely characteristic 4-line ESR spectrum. Using this technique, we have shown that, in addition to Aβ(1–40), Aβ(1–42) and Aβ(25–35), various other amyloidogenic proteins and peptides also have the ability to generate hydrogen peroxide. These include α-synuclein (implicated in PD and related disorders), certain toxic fragments of the PrP (implicated in the transmissible spongiform encephalopathies) and the ABri peptide [responsible for FBD (familial British dementia), see below] [27,31–37]. In all cases where a DMPO-OH spectrum was obtained, its formation could be inhibited by catalase, so confirming the formation of hydrogen peroxide, or by adding a potent metal chelator (e.g. diethylenetriaminepenta-acetic acid) during peptide incubation, suggesting that metal ions are always involved. Numerous controls, including reverse and scrambled peptides, non-toxic peptides and the β- and γ-synucleins were shown to lack any significant activity, when tested under the same experimental conditions, so demonstrating the specificity of hydrogen peroxide formation. Moreover, we have also noted a very good positive correlation between the ability of these various proteins and peptides to generate hydrogen peroxide and their reported toxic effects on cultured cells. An interesting and informative example of the latter is the case of ABri . This peptide is associated with FBD, a rare genetic disorder that is strikingly similar in neuropathology to AD. Affected areas of the brain in FBD have numerous extracellular amyloid plaques and intracellular neurofibrillary tangles. The disorder is due to a stop codon mutation in the BRI gene, the protein product of which undergoes proteolytic cleavage to release an abnormally long peptide fragment (ABri) that rapidly aggregates in vitro into toxic oligomers. In our experiments, an ESR spectrum was only obtained from the oxidized form of ABri (with an intact intramolecular disulphide bond) and no spectra were obtained from the reduced form of this peptide, or from the oxidized or reduced forms of the shorter wild-type peptide . This agrees very closely with reported cell-toxicity data [38,39].
Accumulation of hydrogen peroxide during the early stages of aggregation of Aβ(1–40) (100 μM in PBS)
Hydrogen peroxide is generated during the early stages of protein aggregation
Recently, we have examined how the generation of hydrogen peroxide relates to the extent of peptide aggregation by following the time dependence of hydrogen peroxide formation during incubation of solutions (100 μM in PBS) of Aβ(1–40) and ABri (oxidized form) . We found that, in both the cases, hydrogen peroxide was not generated continuously throughout the aggregation process, but was formed as a short ‘burst’ comparatively early on during the peptide incubation period. AFM (atomic force microscopy) revealed structures resembling ‘protofibrils’ or ‘soluble oligomers’ during these early periods of hydrogen peroxide formation and the total absence of late-stage amyloid fibrils. Mature Aβ fibrils lacked the ability to generate hydrogen peroxide. It should be noted that after prolonged incubation in PBS, at around physiological pH, ABri does not assemble into typical amyloid fibrils, but instead forms ‘amorphous’ deposits that lack Congo Red birefringence and do not bind to thioflavin T [38–40]. This indicates a lack of β-pleated sheet fibrillar structure. The fact that hydrogen peroxide is still generated by ABri under these conditions emphasizes the fact that its formation does not appear to be associated with the presence of mature amyloid fibrils. On the contrary, our data suggest that the toxicity of Aβ and ABri could be due to the generation of hydrogen peroxide by an early form of protein aggregate. In the case of Aβ, this could be explained, at least in part, by the formation of dityrosine cross-links . However, it is also possible that hydrogen peroxide is generated as a ‘by-product’ of the early stages of the aggregation process itself. The latter would be compatible with a recent report closely relating Aβ toxicity to nucleation-dependent protein aggregation . In this report, it was shown that fibrillar Aβ or soluble Aβ alone were not toxic to cultured primary neurons, but when fibrillar Aβ was used to ‘seed’ the polymerization of soluble Aβ, neuronal cell death ensued. In other words, overt neuronal cell death mediated by Aβ was found to be critically dependent on ongoing Aβ aggregation, and not on the presence of any one particular species of neurotoxic aggregate.
The reported neurotoxic effects of various amyloid peptides in vitro could be explained by their ability to generate hydrogen peroxide, and possibly also, as an integral part of this process, to convert suitable reducing substrates (e.g. cholesterol) into highly toxic products. It is now well established that the addition of toxic forms of Aβ and other amyloidogenic peptides to cells causes membrane damage, calcium ion influx, oxidative free radical damage and apopotosis (Figure 2). Furthermore, this toxicity can be inhibited by catalase, free radical scavengers or by antioxidants  and 3-amino-triazole, a catalase inhibitor, enhances the toxic effects of Aβ . This is consistent with exposure of cells to hydrogen peroxide or other ROS. In these toxicity experiments, the amyloidogenic peptides are usually added to cells at low micromolar concentrations. Under suitable conditions, Aβ has been reported to be capable of generating equimolar concentrations of hydrogen peroxide . Hydrogen peroxide is freely permeable across cell membranes, and can induce changes in calcium homoeostasis and apoptosis at submicromolar concentrations . Thus, the amount of this molecule generated from Aβ, or other aggregating proteins, could be sufficient to explain their toxic effects on cultured cells.
Possible common mechanism of cell death in protein aggregation-dependent neurodegenerative diseases
The relevance of the data on Aβ cellular toxicity in vitro to neurodegeneration in vivo is much less certain. However, the focal concentrations of Aβ in the vicinity of early ‘primitive’ senile plaques could easily be expected to reach the low micromolar levels that have been shown to be cytotoxic. Even very low concentrations of hydrogen peroxide, or other ROS, could induce neurodegenerative changes in the brain over the extended time periods involved in the development and progression of AD, and this would also apply to other neurodegenerative diseases. As noted above, there is now substantial evidence indicating that oxidative damage to the brain is one of the earliest pathological events in AD . Our results raise the intriguing possibility that this could be due to the generation of hydrogen peroxide during the early stages of Aβ oligomerization, before the formation of substantial numbers of senile plaques in the brain. In the presence of redox-active metal ions, hydrogen peroxide is readily converted, via the Fenton reaction, into the highly reactive hydroxyl radical, which could be responsible for much of this early oxidative damage. Since oxidative stress can increase the neuronal production of Aβ , the presence of hydrogen peroxide (and the hydroxyl radical) could set up a potentially catastrophic positive feedback mechanism whereby Aβ oligomers stimulate their own production.
It is doubtful whether the redox properties of Aβ, in conjunction with bound metal ions, can explain the potent activity of very low nanomolar concentrations of cell-derived soluble oligomers of Aβ on LTP , but this is not impossible because hydrogen peroxide has been shown to affect synaptic plasticity by acting as a ‘diffusible signalling molecule’ .
The first reports of the generation of hydrogen peroxide from Aβ  have led to clinical trials with clioquinol, a metal ion chelator, as a potential treatment for AD . It is important in the future that many more clinical trials are carried out on drugs that specifically target the generation of hydrogen peroxide during the early stages of protein aggregation, or aim to limit its damaging effects on brain cells, as such drugs could be beneficial in the treatment of several different neurodegenerative diseases. Suitable compounds would include transition metal ion chelators, inhibitors of early-stage oligomer formation, antioxidants, ROS scavengers or drugs that combine more than one of these properties . For maximium potential benefit, these drugs must be given to patients in the early stages of disease.
Proteins in Disease: A Focus Topic at BioScience2005, held at SECC Glasgow, U.K., 17–21 July 2005. Edited by B. Austen (St George's Hospital Medical School, London, U.K.), C. Connolly (Dundee, U.K.), B. Irvine (Belfast, U.K.), M. Sugden (Queen Mary, London, U.K.) and V. Zammit (Hannah Research Institute, Ayr, U.K.).
We thank Michael Goedert (University of Cambridge, Cambridge, U.K.) and David Brown (University of Bath, Bath, U.K.) for samples of synucleins and prion peptides. This work was supported by a Project Grant (GR065764AIA) from The Wellcome Trust.