ß-amyloid fibrils in Alzheimer disease are not inert when bound to copper ions but can degrade hydrogen peroxide and generate reactive oxygen species.

According to the "amyloid cascade" hypothesis of Alzheimer disease, the formation of Aß fibrils and senile plaques in the brain initiates a cascade of events leading to the formation of neurofibrillary tangles, neurodegeneration, and the symptom of dementia. Recently, however, emphasis has shifted away from amyloid fibrils as the predominant toxic form of Aß toward smaller aggregates, referred to as "soluble oligomers." These oligomers have become one of the prime suspects for involvement in the early oxidative damage that is evident in this disease. This raises the question whether or not Aß fibrils are actually "inert tombstones" present at the end of the aggregation process. Here we show that, when Aß(1-42) aggregates, including fibrils, are bound to Cu(II) ions, they retain their redox activity and are able to degrade hydrogen peroxide (H2O2) with the formation of hydroxyl radicals and the consequent oxidation of the peptide (detected by formation of carbonyl groups). We find that this ability increases as the Cu(II):peptide ratio increases and is accompanied by changes in aggregate morphology, as determined by atomic force microscopy. When aggregates are prepared in the copresence of Cu(II) and Zn(II) ions, the ratio of Cu(II):Zn(II) becomes an important factor in the degeneration of H2O2, the formation of carbonyl groups in the peptide, and in aggregate morphology. We believe, therefore, that Aß fibrils can destroy H2O2 and generate damaging hydroxyl radicals and, so, are not necessarily inert end points.

Metal-dependent generation of reactive oxygen species from amyloid proteins implicated in neurodegenerative disease.

Using a method based on ESR spectroscopy and spin-trapping, we have shown that Abeta (amyloid beta-peptide) (implicated in Alzheimer's disease), alpha-synuclein (implicated in Parkinson's disease), ABri (British dementia peptide) (responsible for familial British dementia), certain toxic fragments of the prion protein (implicated in the transmissible spongiform encephalopathies) and the amylin peptide (found in the pancreas in Type 2 diabetes mellitus) all have the common ability to generate H(2)O(2) in vitro. Numerous controls (reverse, scrambled and non-toxic peptides) lacked this property. We have also noted a positive correlation between the ability of the various proteins tested to generate H(2)O(2) and their toxic effects on cultured cells. In the case of Abeta and ABri, we have shown that H(2)O(2) is generated as a short burst during the early stages of aggregation and is associated with the presence of protofibrils or oligomers, rather than mature fibrils. H(2)O(2) is readily converted into the aggressive hydroxyl radical by Fenton chemistry, and this extremely reactive radical could be responsible for much of the oxidative damage seen in all of the above disorders. We suggest that the formation of a redox-active complex involving the relevant amyloidogenic protein and certain transition-metal ions could play an important role in the pathogenesis of several different protein misfolding disorders.

Copper-mediated formation of hydrogen peroxide from the amylin peptide: a novel mechanism for degeneration of islet cells in type-2 diabetes mellitus?

Amyloid deposits derived from the amylin peptide accumulate within pancreatic islet beta-cells in most cases of type-2 diabetes mellitus (T2Dm). Human amylin 'oligomers' are toxic to these cells. Using two different experimental techniques, we found that H(2)O(2) was generated during the aggregation of human amylin into amyloid fibrils. This process was greatly stimulated by Cu(II) ions, and human amylin was retained on a copper affinity column. In contrast, rodent amylin, which is not toxic, failed to generate any H(2)O(2) and did not interact with copper. We conclude that the formation of H(2)O(2) from amylin could contribute to the progressive degeneration of islet cells in T2Dm.

A spectroscopic study of some of the peptidyl radicals formed following hydroxyl radical attack on beta-amyloid and alpha-synuclein.

There is clear evidence implicating oxidative stress in the pathology of many neurodegenerative diseases. Reactive oxygen species (ROS) are the primary mediators of oxidative stress, and hydrogen peroxide, a key ROS, is generated during aggregation of the amyloid proteins associated with some of these diseases. Hydrogen peroxide is catalytically converted to the aggressive hydroxyl radical in the presence of Fe(II) and Cu(I), which renders amyloidogenic proteins such as beta-amyloid and alpha-synuclein (implicated in Alzheimer's disease (AD) and Parkinson's disease (PD), respectively) vulnerable to self-inflicted hydroxyl radical attack. Here, we report some of the peptide-derived radicals, detected by electron spin resonance spectroscopy employing sodium 3,5-dibromo-4-nitrosobenzenesulfonate as a spin-trap, following hydroxyl radical attack on Abeta(1-40), alpha-synuclein and some other related peptides. Significantly, we found that sufficient hydrogen peroxide was self-generated during the early stages of aggregation of Abeta(1-40) to produce detectable peptidyl radicals, on addition of Fe(II). Our results support the hypothesis that oxidative damage to Abeta (and surrounding molecules) in the brain in AD could be due, at least in part, to the self-generation of ROS. A similar mechanism could operate in PD and some other "protein conformational" disorders.

Formation of hydrogen peroxide and hydroxyl radicals from A(beta) and alpha-synuclein as a possible mechanism of cell death in Alzheimer's disease and Parkinson's disease.

The formation of extracellular or intracellular deposits of amyloid-like protein fibrils is a prominent pathological feature of many different neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). In AD, the beta-amyloid peptide (A(beta)) accumulates mainly extracellularly at the center of senile plaques, whereas, in PD, the alpha-synuclein protein accumulates within neurons inside the Lewy bodies and Lewy neurites. We have shown recently that solutions of A(beta) 1-40, A(beta) 1-42, A(beta) 25-35, alpha-synuclein and non-A(beta) component (NAC; residues 61-95 of alpha-synuclein) all liberate hydroxyl radicals upon incubation in vitro followed by the addition of small amounts of Fe(II). These hydroxyl radicals were readily detected by means of electron spin resonance spectroscopy, employing 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent. Hydroxyl radical formation was inhibited by the inclusion of catalase or metal-chelators during A(beta) or alpha-synuclein incubation. Our results suggest that hydrogen peroxide accumulates during the incubation of A(beta) or alpha-synuclein, by a metal-dependent mechanism, and that this is subsequently converted to hydroxyl radicals, on addition of Fe (II), by Fenton's reaction. Consequently, one of the fundamental molecular mechanisms underlying the pathogenesis of cell death in AD and PD, and possibly other neurodegenerative or amyloid diseases, could be the direct production of hydrogen peroxide during formation of the abnormal protein aggregates.

Quinacrine acts as an antioxidant and reduces the toxicity of the prion peptide PrP106-126.

The accumulation of protein aggregates in the brain is a central feature of several different neurodegenerative diseases. We have recently shown that Abeta and alpha-synuclein, associated with Alzheimer's disease, Parkinson's disease and related disorders, can both induce the formation of hydroxyl radicals following incubation in solution, upon addition of Fe(II). PrP106-126, a model peptide for the study of prion protein-mediated cell death, shares the same property. In this study we show that quinacrine (an anti-malarial drug and inhibitor of prion replication) acts as an effective antioxidant, readily scavenging hydroxyl radicals formed from hydrogen peroxide via the Fenton reaction or generated during incubation of the PrP106-126 peptide. Furthermore, the toxicity of PrP106-126 to cultured cells was significantly inhibited by quinacrine.

Copper-dependent generation of hydrogen peroxide from the toxic prion protein fragment PrP106-126.

Oligomeric forms of many of the aggregating proteins associated with neurodegenerative diseases are toxic to cultured cells. We have shown recently that Abeta and alpha-synuclein can both induce the formation of hydroxyl radicals following incubation in solution, upon the addition of Fe(II). Thus, they appear to generate hydrogen peroxide, which is converted to hydroxyl radicals via the Fenton reaction. Here we show that the widely studied toxic peptide fragment of the prion protein, PrP106-126, has exactly the same property, but only in the presence of copper ions. Since the aggregation and toxicity of PrP106-126 have been reported to be critically dependent on copper binding, our data suggest that the published cytotoxic effects of this peptide could also be due to its ability to generate hydrogen peroxide.

The amylin peptide implicated in type 2 diabetes stimulates copper-mediated carbonyl group and ascorbate radical formation.

Human amylin (hA), which is toxic to islet ß-cells, can self-generate H(2)O(2), and this process is greatly enhanced in the presence of Cu(II) ions. Here we show that carbonyl groups, a marker of oxidative modification, were formed in hA incubated in the presence of Cu(II) ions or Cu(II) ions plus H(2)O(2), but not in the presence of H(2)O(2) alone. Furthermore, under similar conditions (i.e., in the presence of both Cu(II) ions and H(2)O(2)), hA also stimulated ascorbate radical formation. The same observations concerning carbonyl group formation were made when the histidine residue (at position 18) in hA was replaced by alanine, indicating that this residue does not play a key role. In complete contrast to hA, rodent amylin, which is nontoxic, does not generate H(2)O(2), and binds Cu(II) ions only weakly, showed none of these properties. We conclude that the hA-Cu(II)/Cu(I) complex is redox active, with electron donation from the peptide reducing the oxidation state of the copper ions. The complex is capable of forming H(2)O(2) from O(2) and can also generate (•)OH via Fenton chemistry. These redox properties of hA can explain its ability to stimulate copper-mediated carbonyl group and ascorbate radical formation. The formation of reactive oxygen species from hA in this way could hold the key to a better understanding of the damaging consequences of amyloid formation within the pancreatic islets of patients with type 2 diabetes mellitus.

Hypothesis: soluble aß oligomers in association with redox-active metal ions are the optimal generators of reactive oxygen species in Alzheimer's disease.

Considerable evidence points to oxidative stress in the brain as an important event in the early stages of Alzheimer's disease (AD). The transition metal ions of Cu, Fe, and Zn are all enriched in the amyloid cores of senile plaques in AD. Those of Cu and Fe are redox active and bind to Aß in vitro. When bound, they can facilitate the reduction of oxygen to hydrogen peroxide, and of the latter to the hydroxyl radical. This radical is very aggressive and can cause considerable oxidative damage. Recent research favours the involvement of small, soluble oligomers as the aggregating species responsible for Aß neurotoxicity. We propose that the generation of reactive oxygen species (i.e., hydrogen peroxide and hydroxyl radicals) by these oligomers, in association with redox-active metal ions, is a key molecular mechanism underlying the pathogenesis of AD and some other neurodegenerative disorders.

Hydrogen peroxide is generated during the very early stages of aggregation of the amyloid peptides implicated in Alzheimer disease and familial British dementia.

Alzheimer disease and familial British dementia are neurodegenerative diseases that are characterized by the presence of numerous amyloid plaques in the brain. These lesions contain fibrillar deposits of the beta-amyloid peptide (Abeta) and the British dementia peptide (ABri), respectively. Both peptides are toxic to cells in culture, and there is increasing evidence that early "soluble oligomers" are the toxic entity rather than mature amyloid fibrils. The molecular mechanisms responsible for this toxicity are not clear, but in the case of Abeta, one prominent hypothesis is that the peptide can induce oxidative damage via the formation of hydrogen peroxide. We have developed a reliable method, employing electron spin resonance spectroscopy in conjunction with the spin-trapping technique, to detect any hydrogen peroxide generated during the incubation of Abeta and other amyloidogenic peptides. Here, we monitored levels of hydrogen peroxide accumulation during different stages of aggregation of Abeta-(1-40) and ABri and found that in both cases it was generated as a short "burst" early on in the aggregation process. Ultrastructural studies with both peptides revealed that structures resembling "soluble oligomers" or "protofibrils" were present during this early phase of hydrogen peroxide formation. Mature amyloid fibrils derived from Abeta-(1-40) did not generate hydrogen peroxide. We conclude that hydrogen peroxide formation during the early stages of protein aggregation may be a common mechanism of cell death in these (and possibly other) neurodegenerative diseases.

Generation of hydrogen peroxide from mutant forms of the prion protein fragment PrP121-231.

By means of electron spin resonance spectroscopy, in conjunction with the spin trapping technique, we have shown previously that Abeta and alpha-synuclein (aggregating proteins that accumulate in the brain in Alzheimer's disease, Parkinson's disease, and related disorders) both induce the formation of hydroxyl radicals following incubation in solution, upon addition of Fe(II). These hydroxyl radicals are apparently formed from hydrogen peroxide, via Fenton's reaction. An N-terminally truncated fragment of the mouse prion protein (termed PrP121-231) is toxic to cerebellar cells in culture, and certain human mutations, responsible for inherited prion disease, enhance this toxicity. Here we report that PrP121-231 containing three such mutations (E200K, D178N, and F198S) also generated hydroxyl radicals, upon addition of Fe(II). The formation of these radicals was blocked by catalase, or by metal chelators, each of which also reduced the toxicity of the PrP121-231 fragments to cultured normal mouse cerebellar cells. Wild-type PrP121-231, full-length cellular PrP, and its homologue doppel did not generate any detectable hydroxyl radicals. We conclude that the additional cytotoxic effects of the mutant forms of PrP121-231 could be due to their ability to generate hydrogen peroxide, by a metal-dependent mechanism. Thus, one effect of these (and possibly other) prion mutations could be production of a particularly toxic form of the prion protein, with an enhanced capacity to induce oxidative damage, neurodegeneration, and cell loss.

Both the D-(+) and L-(-) enantiomers of nicotine inhibit Abeta aggregation and cytotoxicity.

The underlying cause of Alzheimer's disease is thought to be the aggregation of monomeric beta-amyloid (Abeta), through a series of toxic oligomers, which forms the mature amyloid fibrils that accumulate at the center of senile plaques. It has been reported that L-(-)-nicotine prevents Abeta aggregation and toxicity, and inhibits senile plaque formation. Previous NMR studies have suggested that this could be due to the specific binding of L-(-)-nicotine to histidine residues (His6, His13, and His14) in the peptide. Here, we have looked at the effects of both of the L-(-) and D-(+) optical enantiomers of nicotine on the aggregation and cytotoxicity of Abeta(1-40). Surprisingly, both enantiomers inhibited aggregation of the peptide and reduced the toxic effects of the peptide on cells. In NMR studies with Abeta(1-40), both enantiomers of nicotine were seen to interact with the three histidine residues. Overall, our data indicate that nicotine can delay Abeta fibril formation and maintain a population of less toxic Abeta species. This effect cannot be due to a highly specific binding interaction between nicotine and Abeta, as previously thought, but could be due instead to weaker, relatively nonspecific binding, or to the antioxidant or metal chelating properties of nicotine. D-(+)-nicotine, being biologically much less active than L-(-)-nicotine, might be a useful therapeutic agent.