Quantification of Protein Aggregates Using Bimolecular Fluorescence Complementation.

Abstract

Neuronal accumulations of the presynaptic protein α-synuclein represent a hallmark of Parkinson's disease (PD). Multiple system atrophy and dementia with Lewy bodies are other neurodegenerative diseases in which α-synuclein aggregates are the main pathological characteristic. This is why these diseases are summarized as synucleinopathies. The abnormal α-Synuclein accumulation eventually results in the formation of Lewy bodies and Lewy neurites in surviving neurons of the brain. α-Synuclein aggregation has been widely researched in vitro but little is known about α-synuclein aggregation in vivo. This is why it is still unclear whether α-synuclein accumulation and/or which of the resulting aggregates (soluble oligomers or insoluble fibrils) actually trigger neurotoxic events. In our study, we employed the robust genetic makeup of Drosophila melanogaster in combination with bimolecular fluorescence complementation and developed an in vivo assay allowing to determine abundance of soluble α-synuclein aggregates (most likely oligomers). Insoluble α-synuclein aggregates (most likely fibrils) are detected by filter retardation assay. We provide an experimental outline to verify the reported assay system. The described method is easy, inexpensive, fast, and sensitive. Altogether, the described assay system is ideal for large-scale screening approaches aiming to test/identify compounds/conditions that affect α-synuclein aggregation in vivo.Moreover, the experimental outline reported here could serve as a template to analyze aggregation of proteins other than α-synuclein. As accumulation of specific proteins is a hallmark of almost every neurodegenerative disease, only minor changes to our protocol should be necessary for a similar analysis. We have put special focus on critical evaluation steps to consider when adapting our protocol to other aggregate-forming proteins.