Copper-induced structural rearrangements of Abeta40 structure and its redox properties are described in this study. Electrochemical and fluorescent methods are used to characterise the behaviour of Abeta-Cu species. The data suggest that time-dependent folding of Abeta-Cu species may cause changes in the redox potentials.Extracellular deposits of beta-amyloid (Abeta) into senile plaques are the major features observed in brains of Alzheimer's disease (AD) patients. A high concentration of copper has been associated with insoluble amyloid plaques. It is known that Abeta(1-40) can bind copper with high affinity, but electrochemical properties of Abeta(1-40)-Cu complexes are not well-characterised. In this study we demonstrate that complexation of copper (both as Cu(I) and Cu(II)) by Abeta(1-40) reduces the metal electrochemical activity. Formation of copper-Abeta(1-40) complexes is associated with alteration of the redox potential. The data reveal significant redox activity of fresh Abeta-copper solutions. However, copper-induced structural rearrangements of the peptide, documented by CD, correspond with time-dependent changes of formal reduction potentials (E(0')) of the complex. Fluorescent and electrochemical (cyclic voltammetry and differential pulse voltammetry) techniques suggest that reduction of the redox activity by Abeta-Cu complexes could be attributed to conformational changes that diminished copper accessibility to the external environment. According to our evidence, conformational rearrangements, induced by copper binding to amyloid, elongate the time necessary to attain the same beta-sheet content as for the metal-free peptide. Although the redox activity of Abeta-Cu complexes diminishes in a time-dependent manner, they are not completely devoid of toxicity as they destabilize red blood cells osmotic fragility, even after prolonged incubation.

b-Amyloid protein is the major component of senile plaques found in the brains of Alzheimer's patients. Previously, a new biochemical property of amyloid, its ability to disrupt ester and peptide bonds, was described [Elbaum, D., Brzyska, M., Bacia, A. & Alkon, D. (2000) Biochem. Biophys. Res. Commun. 267, 733±738]. In the present work we compare the ability of b-amyloid to hydrolyse and oxidize model fluorescent derivatives of dichlorofluorescein [dichlorodihydrofluorescein (H 2 DCF) or dichlorofluorescein diacetate (DCF-DA), respectively] to the same final product (dichlorofluorescein). Although there is accumulating evidence of oxidative properties of b-amyloid, little is known about its hydrolytic abilities. Chemical modification studies revealed that hydrolytic properties are related to a His, Ser and Asp/Glu triad, while residues of His, Tyr and Met are involved in the oxidative activity of amyloid. Studies with the rat homologue of human b-amyloid (1±40), containing three amino-acid substitutions (Arg53Gly, Tyr103Phe and His133Arg) confirmed a role of His in the studied processes. Reduction of the hydrolysis product caused by inhibitors of Ser esterases (phenylmethylsulphonyl fluoride and eserine) suggests that b-amyloid-mediated hydrolysis is Ser sensitive. Antioxidants and metal chelators that reduced H 2 DCF oxidation did not change or increase DCF-DA hydrolysis. Solvent isotope effects suggest the involvement of hydrogen bonds in the hydrolysis reaction. Hydrolysis was inhibited by redox-active metal ions and was practically oxygen independent while the oxidation process was redox-active-metal enhanced [Cu(II) and Fe(II) primarily], and oxygen dependent. Product formation was significantly inhibited by catalase and superoxide dismutase as well as benzoquinone, a specific superoxide anion radical scavenger. Increase of fluorescence by oxidation was strongly inhibited by azide and His and enhanced in samples prepared with deuterated phosphate buffer, suggesting singlet oxygen intermediacy. Our data are consistent with superoxide-mediated singlet oxygen intermediate in this Fenton mechanism-driven reaction. These results indicate that hydrolytic and oxidative properties of b-amyloid are distinct features of this peptide and probably require different mechanisms to occur, but both of them may contribute to b-amyloid toxicity.Keywords: amino acid; b-amyloid; hydrolysis; metal; oxidation.Alzheimer's disease is a progressive neurodegenerative disorder affecting a large proportion of the ageing population. It is characterized by gradual loss of cognitive function and synaptic integrity, selective neuronal death and the abnormal formation of neuritic and core plaques in the cerebral cortex. The major proteinaceous component of the extracellular amyloid deposits is the b-amyloid peptide, consisting of 39±43 amino acids, which is produced by proteolytic processing of its precursor, the b-amyloid precursor protein (APP). This is a 70-kDa transmembrane protein, expressed in various cell types, including neural cel...

The stress proteins hsp25 and alpha B-crystallin are found in increased concentrations in reactive astrocytes of brains undergoing neurodegeneration. In order to characterize this reaction, we investigated the expression of hsp25 and alpha B-crystallin during growth and after stress (heat shock) in glial cells in vitro. In primary rat brain cultures, hsp25 was present in actively dividing astrocytes that were positive for glial fibrillary acidic protein. alpha B-crystallin was found predominantly in oligodendrocytes. Heat shock resulted in increased concentrations of hsp25 and alpha B-crystallin in astrocytes, without any detectable changes in intracellular localization, as detectable with confocal laser microscopy. These results indicate that a neurodegeneration-related increase of the small stress proteins in astrocytes in independent of gliosis per se, and may be a disease-related event.