Removing or preventing the formation of α-synuclein aggregates is a plausible strategy against Parkinson’s disease. To this end, we have engineered the β-wrapin AS69 to bind monomeric α-synuclein with high affinity. In cultured cells, AS69 reduced the self-interaction of α-synuclein and formation of visible α-synuclein aggregates. In flies, AS69 reduced α-synuclein aggregates and the locomotor deficit resulting from α-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-α-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.

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

Removing or preventing the formation of α-synuclein aggregates is a plausible strategy against Parkinson's disease. To this end we have engineered the β-wrapin AS69 to bind monomeric α-synuclein with high affinity. In cultured cells, AS69 reduced the occurrence of α-synuclein oligomers and of visible α-synuclein aggregates. In flies, AS69 reduced α-synuclein aggregates and the locomotor deficit resulting from α-synuclein expression in neuronal cells. In a mouse model based on the intracerebral injection of pre-formed α-synuclein seed fibrills (PFFs), AS69 co-injection reduced the density of dystrophic neurites observed three months later. In biophysical experiments in vitro, AS69 highly sub-stoichiometrically inhibited autocatalytic secondary nucleation processes, even in the presence of a large excess of monomer.We present evidence that the AS69-α-synuclein complex, rather than the free AS69, is the inhibitory species responsible for sub-stoichiometric inhibition. These results represent a new paradigm that high affinity monomer binders can be strongly sub-stoichiometric inhibitors of nucleation processes.

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