Abstract
The accumulation of amyloid β peptide(1-42) (Aβ(1-42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of Aβ(1-42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. Aβ may be present in an aggregated form prior to cellular uptake. Alternatively, monomeric peptide may enter the endocytic pathway and conditions in the endocytic compartments may induce the aggregation process. Our study aims to answer the question whether aggregate formation is a prerequisite or a consequence of Aβ endocytosis. We visualized aggregate formation of fluorescently labeled Aβ(1-42) and tracked its internalization by human neuroblastoma cells and neurons. β-Sheet-rich Aβ(1-42) aggregates entered the cells at low nanomolar concentration of Aβ(1-42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed Aβ(1-42) aggregates to form. By uncoupling membrane binding from internalization, we found that Aβ(1-42) monomers bound rapidly to the plasma membrane and formed aggregates there. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with metabolic inhibition. Our data therefore imply that the formation of β-sheet-rich aggregates is a prerequisite for Aβ(1-42) uptake and cytotoxicity.
Highlights
One of the pathological hallmarks of Alzheimer disease (AD)[2] is the presence of extracellular plaques composed mainly of 42-amino acid amyloid  peptide (A(1– 42)) (1)
By uncoupling membrane binding from internalization, we found that A(1– 42) monomers bound rapidly to the plasma membrane and formed aggregates there
We found that cultured human neuroblastoma cells (SH-EP cells) can efficiently internalize -sheet-rich aggregates of A(1– 42) at nanomolar concentrations, and that cellular uptake was associated with metabolic inhibition
Summary
One of the pathological hallmarks of Alzheimer disease (AD)[2] is the presence of extracellular plaques composed mainly of 42-amino acid amyloid  peptide (A(1– 42)) (1). Our understanding of the connection between aggregation and cytotoxicity is still limited. It has not been conclusively determined how and when the A(1–42) peptide becomes toxic, whether A aggregates prior to internalization or during the internalization process and, if so, in which intracellular compartments the aggregates form. Elucidating the connection between aggregation and internalization of A(1–42) peptide may be vital in understanding its toxicity. We aimed to determine whether the formation of aggregates and -sheet-rich structures, as reported by thioflavin dyes (20) and conformation-specific antibodies is a prerequisite for its neuronal uptake. We found that cultured human neuroblastoma cells (SH-EP cells) can efficiently internalize -sheet-rich aggregates of A(1– 42) at nanomolar concentrations, and that cellular uptake was associated with metabolic inhibition. Internalization occurred after aggregation on the plasma membrane, which suggests that the membrane is an environment that facilitates aggregation of A(1–42) monomers
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