(53d) Investigation of the Residue Specific Interactions of Aβ with Neuron-Like Cell Surface Molecules: Implications for the Mechanism of Pathogenesis of Alzheimer's Disease

Alzheimer's disease (AD) is a progressive neuro-degenerative disorder. Memory impairment, as well as problems with language, decision-making ability, judgment, and personality are the various symptoms of this disease. AD primarily affects people over 65 years of age. In view of the United States’ ageing population, it is especially important to discover new therapeutic approaches to combat this disease.

Senile plaques and neuro-fibrillary tangles are observed in the brains of AD patients at the histopathological level. Beta-amyloid (Aβ) peptide is a major component of these senile plaques. Aggregated Aβ is believed to be toxic to neurons and is implicated in Alzheimer’s disease. However, the mechanism by which Aβ leads to neurodegeneration is still unclear.  Elucidating steps in the mechanism of Aβ-neuron interaction that leads to AD pathology may provide insight into new therapeutic avenues to treat AD.

The neurotoxicity of Aβ is thought to be dependent upon its spontaneous adoption of an aggregated structure.  A number of residues in the beta amyloid peptide have been found to be linked to peptide neurtoxicity, including Methoionine 35 (M35), residues 16-22, Glycine 33 (G33) and Asparagine 27 (N27).  Some of these residues like M35 and G33 has specifically been linked to Aβ toxicity via their oxidative potential or their affect on neuron electrical activity (long term potentiation or LTP), respectively .  Other residues such as region 17-21 are thought to be important because of the role of this hydrophobic region in Aβ aggregation.

We hypothesize that the sequence of Aβ has a role to play in its aggregated structure, its association with cells and its resultant toxicity. We test this hypothesis by analyzing if particular residues of the structure of Aβ are important for binding to cell components and for inducing changes in cell signaling linked to Aβ pathology.

We used fluorescence resonance energy transfer (FRET) and confocal microscopy, along with flow cytometry of both mutated and chemically modified Aβ-40 sequences to probe the importance of different residues on Aβ in the interaction of aggregated Aβ with neuron-like cells.  Initially we showed that Aβ bound near an integrin receptor (α6) on the cell surface. The first 7 residues of Aβ contain a possible integrin binding sequence, RHD, thus we examined the possibility that these residues were important in Aβ-40 interaction. Results suggest that the 8-40 mutant of Aβ-40 , has significantly higher energy transfer to the integrin than wild type Aβ40 leading to the speculation , that the 1-7 residues and the RHD sequence that occurs in 1-8 residue sequence are not crucial for the binding of Aβ-40 to integrin.  Further, we show that methylation of lysines on the Aβ sequence (K16 and K28), and mutation of N27 to proline significantly decreased FRET between Aβ and the α6 integrin receptor. Our results suggest that the sequence of beta amyloid has an important role to play in how it binds to cellular molecules, possibly in turn influencing signaling events in the cell.

Given our results to date, we believe that it is probable that the binding of the aggregated Aβ peptide to the cell is regulated by electrostatic interactions.  We also have multiple lines of evidence that residues 27-33 may be important in Aβ cell interaction.  We will continue to investigate these hypotheses using both mutated and chemically modified Aβ.

Our FRET studies suggest Aβ binds near the α6 integrin receptor subunit.  We also have demonstrated that Aβ activates a tyrosine kinase (possibly from the Src family) upon interaction with the neuron-like cell.   It is possible that activation of the integrin receptor, followed by tyrosine kinase activation, could lead to cellular changes that may impact LTP or other molecular components of neuron learning and memory.  We will present our data showing how the same residue specific changes in Aβ that affect energy transfer with the integrin receptor alter tyrosine kinase activity and our understanding of the mechanism of this signaling cascade in cells exposed to aggregated Aβ.