One of the key pathologic findings of Alzheimer's disease (AD) is senile (amyloid) plaques, composed of aggregated amyloid surrounded by dystrophic neurites and other moieties. Beta-amyloid (Abeta) is a 39-43 amino acid peptide that is toxic to neurons only after an """"""""aging"""""""" period. In contrast, the 11 amino acid fragment of Abeta containing residues 25- 35, Abeta (25-35), is highly toxic immediately upon solubilization. Previously, we reported strong evidence for an O2-dependent, but metal- independent, free-radical-based mechanism for abeta aggregation and neurotoxicity. Abeta generates free radicals after an """"""""aging"""""""" period while Abeta(25-35) developed free radicals immediately upon solubilization. We developed a model to explain the neurotoxicity of abeta to neuronal and glial membranes and have provided experimental evidence to support this model. A free radical-based mechanism of AD is consistent with the myriad of membrane protein and lipid alterations reported in AD. Insight into the mechanism by which these Abeta-induced free radicals arise is needed. The proposed research will provide this insight.
In Specific Aim # 1, we will define systematically the compositional and structural determinants of Abeta(25-35) that lead to free radical production and neurotoxicity. The hypothesis to be tested is that key amino acids in, and the structure of, Abeta peptides are essential to their free radical generating nd neurotoxic properties. It is unclear whether the PBN-trapped radical is oxygen-centered or carbon- centered. Consequently, in Specific Aim # 2, we will use 17O2, which has a nuclear spin, to determine this point.
In Specific Aim # 3, based on insights gained in Specific Aims # 1 and 2, we will use specific substitutions of key amino acids in Abeta(1-40) to assess their importance in the free radical generating and neurotoxic properties of Abeta(1-40).
In Specific Aim # 4, we will use EPR to test a prediction of the model we developed, namely, that Abeta(1-40) [and more rapidly Abeta(25-35)] will produce membrane damage to critical components of cortical neuronal and glial membranes. The hypothesis to be tested is that Abeta(1-40), Abeta(25-35), and Abeta peptides produced from Specific Aims 1-3, that produce a 3-line EPR spectrum with the spin trap PBN, will produce regional damage within the neocortical synaptosomal membrane. In each Aim, we will correlate EPR results with Abeta peptide-induced neurotoxicity, reactive oxygen species production, protein oxidation, Ca2+ flux, glutamate uptake, changes in peptide structure assessed by CD, amino acid alterations (e.g., met sulfoxide formation), the ability to form fibrils as assessed by EM, and abrogation of these free radical effects by appropriate free radical scavengers. Insight into the molecular basis of AD and potential therapeutic intervention is envisaged.
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