Structural and Kinetic Characterization of Cell Surface and Internalized Alzheimer Amyloid Peptides in Neuronal Cells

Alzheimer’s disease is linked to the formation of amyloid fibrils, which are primarily composed of two Alzheimer amyloid peptides, Abeta40 and Abeta42. The peptides start to deposit in brains as plasma membrane-bound diffuse plaques. Current detection methods utilize dyes or antibodies that bind specific conformations of the peptides. However, these observation tools are limited, as they fail to detect the whole ensemble of pre-amyloid structural conformations. We adopted the approach of covalently attaching a fluorescent molecule to the N-terminus of Abeta, which allows for following the aggregation profile and examination of the association of Abeta with neuronal cells lines in real time. With the aid of confocal microscopy and flow cytometry, the rate of Abeta association to neuronal cell lines was found to correlate directly with their aggregation propensities. A non-aggregating mutant of Abeta42 did not bind to live cells and none of these peptides were found to associate with a non-neuronal human lymphoma cell line, U937, which is resistant to Abeta toxicity. Aggregation of Abeta42 on the surface of cells was characterized over time using photobleaching Forster resonance energy transfer, fluorescence quenching, and photobleaching recovery. Furthermore, exposed regions of Abeta aggregates on the cell surface were identified with sequence-specific antibodies. Two populations of aggregates were revealed; the first population displayed reduced energy transfer, showed fluorescence quenching, and bound antibodies specific for the C-terminal of Abeta, whereas the second population of aggregates was capable of energy transfer, was resistant to quenching, and increased in number over time. Interestingly, neither population of aggregates displayed photobleaching recovery. Addition of Abeta monomers to neuronal cells lead to the formation of cell surface aggregates that were eventually internalized into endosomes and lysosomes. The rate of internalization was greatly enhanced when the peptides were partially aggregated by exposure to conditions similar to the lumen of endosomes. Highly aggregated Abeta did not bind to neuronal cells. These studies have mapped out the aggregation pathway of Abeta in the physiologically relevant milieu of neuronal cells in culture, and have potentially revealed information about the formation of the pathological hallmark of Alzheimer’s disease, the senile plaque.Ph