In Vivo Protein Complementation Demonstrates Presynaptic α-Synuclein Oligomerization and Age-Dependent Accumulation of 8–16-mer Oligomer Species

Abstract

Intracellular accumulation of a-synuclein (a-syn) and formation of Lewy bodies are neuropathological characteristics of Parkinson's disease (PD) and related a-synucleinopathies. Oligomerization and spreading of a-syn from neuron to neuron have been suggested as key events contributing to the progression of PD. To directly visualize and characterize a-syn oligomerization and spreading in vivo, we generated two independent conditional transgenic mouse models based on a-syn protein complementation assays using neuron-specifically expressed split Gaussia luciferase or split Venus yellow fluorescent protein (YFP). These transgenic mice allow direct assessment of the quantity and subcellular distribution of a-syn oligomers in vivo. Using these mouse models, we demonstrate an age-dependent accumulation of a specific subtype of a-syn oligomers. We provide in vivo evidence that, although a-syn is found throughout neurons, a-syn oligomerization takes place at the presynapse. Furthermore, our mouse models provide strong evidence for a transsynaptic cell-to-cell transfer of de novo generated a-syn oligomers in vivo.

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

“…Together, these observations combined with previous findings from independent groups demonstrate that the a-syn-Venus-BiFC system does not reveal known differences in the oligomerization and aggregation propensities of PD-associated variants or the interactions between mutants and WT a-syn (Eckermann et al, 2015). Finally, consistent with previous reports, we showed that overexpression of either Vn-a-syn or the two BiFC fragments results in the formation of a-syn oligomers that do not seem to convert to inclusions or amyloid-like aggregates (Dettmer et al, 2015;Gonçalves et al, 2016;Kiechle et al, 2019;Lázaro et al, 2014;Moussaud et al, 2015;Outeiro et al, 2008). Together, these findings underscore the critical importance of characterizing BiFC-based cellular assays at the biochemical and structural levels and understanding how the fusion of fragments of fluorescent proteins influences a-syn biophysical and cellular properties before using such assays in mechanistic, target validation and drug discovery studies.…”

“…This is especially important when the fluorescence signal arises from only a small fraction of the total a-syn expressed in the cell. Finally, whether the oligomers formed by Vn-a-syn could influence a-syn cellular properties in cellular assays using Venus-BiFC system remains unknown (Kiechle et al, 2019;Zondler et al, 2014). Together, these observations underscore the limitations of relying solely on the fluorescence signal as the primary readout of a-syn oligomerization.…”

“…Several a-syn BiFC cellular assays (Gonçalves et al, 2010) have been developed and are increasingly used in cellular and animal studies aimed at identifying and validating the targets or mechanisms of a-syn aggregation, propagation and toxicity. These assays have also been used for screening genetic, enzymatic and pharmacological modulators of a-syn aggregation, toxicity and clearance (Cai et al, 2018;Dimant et al, 2013;Eastwood, Baker, Brooker, Frank, & Mulvihill, 2017;Kiechle et al, 2019;Moussaud et al, 2015;Outeiro, Putcha, Tetzlaff, Spoelgen, Koker, Carvalho, et al, 2008;Prasad et al, 2018). Despite this, there has been very little effort to validate these assays (Eckermann et al, 2015) at the biochemical level and to characterize the nature of the a-syn oligomers that form upon reconstitution of the a-syn BiFC fragments and fluorescence signal.…”

“…the aggregates may consist purely of the aggregation-prone fragment Vn-a-syn or in complex with a-syn-Vc). In a further study, Kiechle et al investigated age-dependent changes to a-syn oligomerization and aggregation at the synapse in two transgenic mouse models where a-syn was fused to the N-or C-terminal part of hGaussia Luciferase or YFP Venus halves (Kiechle et al, 2019). For the hGaussia Luciferase protein-fragment system, the two complementation fragments were expressed at different levels but for the Venus system, the author presented data that showed the two BiFC fragments both equally and differentially expressed.…”

“…The a-syn-BiFC system has been used to develop cellular assays to investigate several aspects of a-syn aggregation and biology, including 1) investigating the molecular and sequence determinants of a-syn oligomerization and aggregation (Aelvoet et al, 2014;Bae et al, 2014;K M Danzer et al, 2011;Karin M. Danzer et al, 2012;Delenclos et al, 2016;Gonçalves et al, 2016;Putcha et al, 2010;Savolainen et al, 2015;Tetzlaff et al, 2008;Zheng et al, 2018); 2) a-syn cell-to-cell transmission and propagation (Bae et al, 2014;Kim et al, 2016); 3) the screening of genetic (Dettmer et al, 2015;Lázaro et al, 2016Lázaro et al, , 2014 and pharmacological modifiers (Dominguez-Meijide et al, 2020;Mohammadiet al, 2017;Moussaud et al, 2015) of a-syn oligomerization, aggregation and clearance; and 4) the validation of therapeutic targets (Outeiro et al, 2008). This approach was also extended to animal models of synucleinopathies, such as rats (Dimant et al, 2013), C. elegans (Kim et al, 2016) and, most recently, Drosophila (Prasad et al, 2018) and mice (Cai et al, 2018) (Kiechle et al, 2019).…”