Fast Fluorescence Lifetime Imaging Reveals the Aggregation Processes of α-Synuclein and Polyglutamine in Aging Caenorhabditis elegans

Abstract

The nematode worm Caenorhabditis elegans has emerged as an important model organism to study the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system, however, remains a challenge. To address this problem, we here present a fast time-gated fluorescence lifetime imaging (TG-FLIM) method and show that it provides functional insights into the process of protein aggregation in living animals by enabling the rapid characterisation of different types of aggregates. More specifically, in longitudinal studies of C. elegans models of Parkinson's and Huntington's diseases, we observed marked differences in the aggregation kinetics and the nature of the protein inclusions formed by α-synuclein and polyglutamine. In particular, we found that α-synuclein inclusions do not display amyloid-like features until late in the life of the worms, whereas polyglutamine forms amyloid characteristics rapidly in early adulthood. Furthermore, we show that the TG-FLIM method is capable of imaging live and non-anaesthetised worms moving in specially designed agarose micro-chambers. Taken together, our results show that the TG-FLIM method enables high-throughput functional imaging of living C. elegans that can be used to study in vivo mechanisms of aggregation and that has the potential to aid the search for therapeutic modifiers of protein aggregation and toxicity.

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

“…In line with previous reports [47], we observed age-dependent coalescence of a-synuclein-YFP into distinct cytoplasmic inclusions ( Figure 1A). Previous fluorescence recovery after photobleaching (FRAP) and FLIM studies have suggested that asynuclein in inclusions exists in a mobile, non-amyloid state [44][45][46][47]. Here, we performed high resolution FLIM to be able to differentiate the protein localised at inclusions from the diffuse (non-aggregated) pool (Figure 1A).…”

“…To understand the initial events in the a-synuclein aggregation process, we assessed its selfassociation using confocal fluorescence lifetime imaging (FLIM) in the nematode worm Caenorhabditis elegans. The fluorescence lifetime of a fluorophore has been previously shown to be an accurate measure of protein self-assembly and amyloid formation, independent of protein concentration or fluorescence intensity [44][45][46]. Given the optical transparency and well-established genetics of C. elegans, we sought to compare the fluorescence lifetime of a human a-synuclein-YFP fusion protein expressed in body wall muscle cells (OW40 worm model, see Methods).…”

Biophysical studies of protein misfolding and aggregation in in vivo models of Alzheimer's and Parkinson's diseases

“…Furthermore, the use of biosensors in combination with fluorescence microscopy may shed more light on the conformational state of protein aggregates in vivo , as demonstrated e.g. by the changes in the fluorescence lifetime of protein tags upon amyloid formation (Kaminski Schierle et al ., 2011 a ; Laine et al ., 2019).…”

“…Whether phase separation drives tau aggregation in human disease remains to be determined, and it will be exciting to study this phenomenon in in vivo models, e.g. by using fluorescence lifetime imaging microscopy (FLIM, Table 1) in Caenorhabditis elegans (Laine et al ., 2019) (see Section ‘α-Synuclein models’).…”

“…Interestingly, fluorescence recovery after photobleaching (FRAP) measurements (Table 1) have indicated that α-synuclein remains mobile within the inclusions, and only in old worms (from day 11 onwards) immobile inclusions, indicative of aggregated proteins, occur (van Ham et al ., 2008). These results were confirmed recently by FLIM (Table 1), in which reduction of the fluorescence lifetime of the YFP tag, indicative of fibril formation by α-synuclein, is only observed after day 10 (Laine et al ., 2019). However, motility defects are observed from day 4 onwards (van Ham et al ., 2008), suggesting that inclusion formation generates toxicity prior to the formation of detectable amyloid-like aggregates.…”