Monitoring α‐synuclein multimerization in vivo

Abstract

The pathophysiology of Parkinson's disease is characterized by the abnormal accumulation of α‐synuclein (α‐Syn), eventually resulting in the formation of Lewy bodies and neurites in surviving neurons in the brain. Although α‐Syn aggregation has been extensively studied in vitro, there is limited in vivo knowledge on α‐Syn aggregation. Here, we used the powerful genetics of Drosophila melanogaster and developed an in vivo assay to monitor α‐Syn accumulation by using a bimolecular fluorescence complementation assay. We found that both genetic and pharmacologic manipulations affected α‐Syn accumulation. Interestingly, we also found that alterations in the cellular protein degradation mechanisms strongly influenced α‐Syn accumulation. Administration of compounds identified as risk factors for Parkinson's disease, such as rotenone or heavy metal ions, had only mild or even no impact on α‐Syn accumulation in vivo. Finally, we show that increasing phosphorylation of α‐Syn at serine 129 enhances the accumulation and toxicity of α‐Syn. Altogether, our study establishes a novel model to study α‐Syn accumulation and illustrates the complexity of manipulating proteostasis in vivo.—Prasad, V., Wasser, Y., Hans, F., Goswami, A., Katona, I., Outeiro, T. F., Kahle, P. J., Schulz, J. B., Voigt, A. Monitoring α‐synuclein multimerization in vivo. FASEB J. 33, 2116–2131 (2019). http://www.fasebj.org

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

“…2E). Surprisingly, this imbalance has been repeatedly observed but not discussed in previous studies (Cai et al, 2018;Dominguez-Meijide et al, 2020;Eckermann et al, 2015;Gustafsson et al, 2018;Lázaro et al, 2016Lázaro et al, , 2014Prasad et al, 2018;Zondler et al, 2014).…”

“…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 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).…”

Protein Quality Control Pathways at the Crossroad of Synucleinopathies

“…However, it is still neither clear whether such assemblies are of fibrillar nature, as the interaction of little as two ␣-Syn molecules is already sufficient to reconstitute fluorescence, nor to what extent the reconstitution of the functional fluorescent protein drives assembly. Nevertheless, some degree of ␣-Syn fibrillation was detected upon overexpression of distinct split Venus-␣-Syn in flies [68]. In any case, true detergentinsoluble ␣-Syn aggregates are either usually not observed in unseeded ␣-Syn models, or they comprise only a small fraction of the total ␣-Syn pool, highlighting the urgent need for better models to document ␣-Syn aggregation.…”

“…Others have employed the co-expression of ␣-Syn with distinct aggregationprone proteins that co-localize with ␣-Syn in LBs to trigger inclusion formation, such as synphilin-1 [45,[62][63][64][65] and tubulin polymerization-promoting protein (TPPP/p25␣) [66], but it is not entirely clear whether these are indeed active drivers of ␣-Syn aggregation. Some studies have also used bimolecular fluorescence complementation assays to assess de novo ␣-Syn aggregation [45,67,68]. In these cases, fluorescence is reconstituted and detected upon coexpression of two ␣-Syn constructs fused to either the N-or C-terminus halves of a fluorescent protein (for example, the split Venus-system).…”