A FRET Sensor for Non‐Invasive Imaging of Amyloid Formation in Vivo

Abstract

Misfolding and aggregation of amyloidogenic polypeptides lie at the root of many neurodegenerative diseases. Whilst protein aggregation can be readily studied in vitro by established biophysical techniques, direct observation of the nature and kinetics of aggregation processes taking place in vivo is much more challenging. We describe here, however, a Förster resonance energy transfer sensor that permits the aggregation kinetics of amyloidogenic proteins to be quantified in living systems by exploiting our observation that amyloid assemblies can act as energy acceptors for variants of fluorescent proteins. The observed lifetime reduction can be attributed to fluorescence energy transfer to intrinsic energy states associated with the growing amyloid species. Indeed, for α-synuclein, a protein whose aggregation is linked to Parkinson's disease, we have used this sensor to follow the kinetics of the self-association reactions taking place in vitro and in vivo and to reveal the nature of the ensuing aggregated species. Experiments were conducted in vitro, in cells in culture and in living Caenorhabditis elegans. For the latter the readout correlates directly with the appearance of a toxic phenotype. The ability to measure the appearance and development of pathogenic amyloid species in a living animal and the ability to relate such data to similar processes observed in vitro provides a powerful new tool in the study of the pathology of the family of misfolding disorders. Our study confirms the importance of the molecular environment in which aggregation reactions take place, highlighting similarities as well Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts as differences between the processes occurring in vitro and in vivo, and their significance for defining the molecular physiology of the diseases with which they are associated.

Dynamic Imaging by Fluorescence Correlation Spectroscopy Identifies Diverse Populations of Polyglutamine Oligomers Formed in Vivo

“…We did not directly relate the brightness to the number of Qn-YFP present in a given species as there is a non-linear relationship between brightness and the number of YFP molecules present in an oligomer. One proposed reason for this is that hetero-FRET occurs upon oligomerization and aggregation, resulting in a decreased fluorescence lifetime (58). Taken together, the brightness and D analysis reveal that while the oligomers increase in both size and brightness as the Q35 animals age, they still maintain a heterogeneous distribution from day 2 to day 6 consistent with the conclusion that specific oligomeric species do not accumulate and that the population as a whole shifts toward larger oligomers (Fig.…”

Cytosolic NADH-NAD+Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging

“…Most sensors that are used by excitation ratio measurement do not have a substantial change in fluorescence lifetime [e.g., (13,25)], as the fluorescence intensity change is predominantly a change between different absorbance states that produce the same, or similar excited states. Most sensors designed to exploit Förster resonance energy transfer (FRET) between two different FPs do show a change in fluorescence lifetime of the ''donor'' FP, as FRET provides an additional nonradiative pathway out of the excited state and thus speeds its decay [e.g., (10,14,15)]. …”

Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments

“…The ease and simplicity with which the tunability is achieved over such a large range is a distinct advantage of this approach. The optical pulse of these supercontinuum sources is around 10 ps (Kaminski-Schierle et al, 2011), they are now commercially available and have been used for FLIM Kaminski-Schierle et al, 2011;McConnell et al, 2007).…”