Mechanisms of cellular binding and internalisation of the [Beta]-amyloid protein of Alzheimer's disease.

Alzheimer's disease (AD) is characterised by an accumulation of the β-amyloid (Aβ) in the brain. Aβ-induced neuronal dysfunction in AD is probably mediated via a direct interaction with components of the neuronal cell membrane. Genetic and molecular evidence suggests that cholesterol and lipoprotein homeostasis influence AD pathogenesis, however the mechanism by which this occurs is unclear. The present study aimed to examine whether cholesterol or lipoprotein factors regulate the binding of Aβ to neuronal cells. To examine Aβ-cell binding, an N-terminal fluorescein (Fluo) conjugate of the 42-amino acid isoform of Aβ (Aβ1-42; FluoAβ1-42) was incubated with SH-SY5Y human neuroblastoma cells, and cell-¬associated fluorescence was measured by confocal microscopy and flow cytometry. FluoAβ1-42 bound to cells in an aggregation-dependent manner, and was internalised to late endocytic compartments. The cell binding and uptake of FluoAβ1-42 was not mediated by binding sites for either cholera toxin B subunit, or antibodies to the low-density lipoprotein-related protein 1 (LRPl). These data suggested that FluoAβ1-42 did not bind to GMl gangliosides or LRPI on the cell surface. However, FluoAβ1-42 did colocalise to the binding sites of the LRPI ligand, receptor-associated protein (RAP), on the plasma membrane. Moreover, co¬incubation with RAP enhanced the binding of Aβ to cells, and Aβ was immunoprecipitated by an anti-RAP antibody following the incubation of RAP with Aβ in vitro. By SDS-PAGE, it was also observed that RAP inhibited the oligomerisation of Aβ, and formed an SDS-stable complex with Aβ. An inhibition of Aβ aggregation was also noted by atomic force microscopy. Since Aβ aggregation alters its toxicity, the effect of RAP on Aβ-induced neurotoxicity was tested. RAP inhibited both the Aβ-induced increase in intracellular calcium of SH-SY5Y cells, and Aβ-induced amnesia in chicks. Since the aggregation of Aβ into SDS-stable low molecular weight oligomers has been closely linked to Aβ neurotoxicity and AD, the effect of RAP on Aβ oligomerisation was examined in more detail. RAP inhibited the formation of SDS-stable dimers of Aβ. The binding and uptake of Aβ and RAP-Aβ by SH-SY5Y cells was inhibited by heparin. However, heparin did not inhibit the formation of the RAP-Aβ complex, suggesting that the binding of Aβ to cells involves its heparin-binding domains, whereas the binding of Aβ to RAP does not. Together, these findings suggest that RAP alters Aβ aggregation, cellular uptake of Aβ, and Aβ-induced neuronal dysfunction. Therefore, the RAP-Aβ interaction may be a viable target for the development of AD therapies.