Ultrasensitive Measurement of Ca2+ Influx into Lipid Vesicles Induced by Protein Aggregates.

Patrick Flagmeier,Suman De,Serge Muyldermans,David Klenerman,Sonia Gandhi,Tuomas P J Knowles,David C Wirthensohn,Cécile Vincke,Steven F Lee,Christopher M Dobson

doi:10.1002/anie.201700966

Abstract

To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high‐throughput assay based on measuring the extent of aggregate‐induced Ca2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aβ42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aβ peptide. We show that the assay can be used to study aggregates from other proteins, such as α‐synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.

Highlights

To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca2+ entry into individual lipid vesicles has been developed
This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy
Picomolar concentrations of Ab42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Ab peptide

Summary

Introduction

To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca2+ entry into individual lipid vesicles has been developed. Methods to detect membrane disruption in bulk solution, based on measuring dye leakage, are valuable for high mm concentrations of toxic species,[20] but we utilize nanosized vesicles filled with a dye whose fluorescence increases on binding Ca2+ ions.

Results

Conclusion