Multiparametric fluorescence microscopy imaging of amyloid beta allows for direct observations of protein misfolding in vitro and in cells

ELIN K. ESBJORNER; CARLOS W. BERTONCINI; SIMON SCHLACHTER; CHRISTOPHER M. DOBSON; CLEMENS F. KAMINSKI; GABRIELE S. KAMINSKI SCHIERLE

Gothenburg

Congreso; 35th Congress of the Federation of European Biochemical Societies; 2010

Misfolding and aggregation of amyloid beta peptides into oligomers and subsequently amyloid fibrils have detrimental effects on synapse function and cell viability, accounting in part for the memory loss and neuronal degeneration associated with Alzheimer´s disease. Whilst amyloid beta aggregation can be readily studied in vitro by established biophysical techniques, it has to date, been very difficult to directly monitor aggregation or identify the aggregation state of misfolded species in vivo with both spatial and temporal resolution. Multiparametric fluorescence microscopy allows for pixel-by-pixel acquisition of not only fluorescence intensities, but also of more responsive parameters such as fluorophore excited state lifetime and steady-state anisotropy and we have recently shown how these parameters sensitively report on the aggregation state of an engineered alpha-synuclein fusion protein with a c-terminal yellow fluorescent protein tag in vitro, in cells and in vivo. Here we have studied misfolding of amyloid beta (Ab40 and Ab42) in vitro and in cultures of neuronal cells. Cross-validation of multiparametric imaging data obtained in vitro with fluorescence spectroscopy, transmission electron microscopy and biochemical assays has allowed us to correlate certain fluorescence lifetime signatures and fluorescence anisotropy characteristics with the specific appearance of monomeric, oligomeric and fibrillar amyloid beta in our samples. Using this information we have then been able to identify the same species in a cellular context by examining the extracellular appearance, internalization and in-cell compartmentalization of Ab40 and Ab42.