An engineered monomer binding-protein for α-synuclein efficiently inhibits the proliferation of amyloid fibrils.

Removing or preventing the formation of [Formula: see text]-synuclein aggregates is a plausible strategy against Parkinson's disease. To this end, we have engineered the [Formula: see text]-wrapin AS69 to bind monomeric [Formula: see text]-synuclein with high affinity. In cultured cells, AS69 reduced the self-interaction of [Formula: see text]-synuclein and formation of visible [Formula: see text]-synuclein aggregates. In flies, AS69 reduced [Formula: see text]-synuclein aggregates and the locomotor deficit resulting from [Formula: see text]-synuclein expression in neuronal cells. In biophysical experiments in vitro, AS69 highly sub-stoichiometrically inhibited both primary and autocatalytic secondary nucleation processes, even in the presence of a large excess of monomer. We present evidence that the AS69-[Formula: see text]-synuclein complex, rather than the free AS69, is the inhibitory species responsible for sub-stoichiometric inhibition of secondary nucleation. These results represent a new paradigm that high affinity monomer binders can lead to strongly sub-stoichiometric inhibition of nucleation processes.

Quantification of Protein Aggregates Using Bimolecular Fluorescence Complementation.

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.

Monitoring α-synuclein multimerization in vivo.

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.