RESUMEN
Multiple isoforms of aggregation-prone proteins are present under physiological conditions and have the propensity to assemble into co-oligomers with different properties from self-oligomers, but this process has not been quantitatively studied to date. We have investigated the amyloid-ß (Aß) peptide, associated with Alzheimer's disease, and the aggregation of its two major isoforms, Aß40 and Aß42, using a statistical mechanical modelling approach in combination with in vitro single-molecule fluorescence measurements. We find that at low concentrations of Aß, corresponding to its physiological abundance, there is little free energy penalty in forming co-oligomers, suggesting that the formation of both self-oligomers and co-oligomers is possible under these conditions. Our model is used to predict the oligomer concentration and size at physiological concentrations of Aß and suggests the mechanisms by which the ratio of Aß42 to Aß40 can affect cell toxicity. An increased ratio of Aß42 to Aß40 raises the fraction of oligomers containing Aß42, which can increase the hydrophobicity of the oligomers and thus promote deleterious binding to the cell membrane and increase neuronal damage. Our results suggest that co-oligomers are a common form of aggregate when Aß isoforms are present in solution and may potentially play a significant role in Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/química , Modelos Químicos , Fragmentos de Péptidos/química , Multimerización de Proteína , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Humanos , Fragmentos de Péptidos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
Local delivery of amyloid beta oligomers from the tip of a nanopipette, controlled over the cell surface, has been used to deliver physiological picomolar oligomer concentrations to primary astrocytes or neurons. Calcium influx was observed when as few as 2000 oligomers were delivered to the cell surface. When the dosing of oligomers was stopped the intracellular calcium returned to basal levels or below. Calcium influx was prevented by the presence in the pipette of the extracellular chaperone clusterin, which is known to selectively bind oligomers, and by the presence a specific nanobody to amyloid beta. These data are consistent with individual oligomers larger than trimers inducing calcium entry as they cross the cell membrane, a result supported by imaging experiments in bilayers, and suggest that the initial molecular event that leads to neuronal damage does not involve any cellular receptors, in contrast to work performed at much higher oligomer concentrations.