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

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.

Keywords: Alzheimer's disease; fluorescence imaging; nano-scale biophysics; neurodegeneration; protein aggregation.