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bioRxiv · 10.1101/2024.02.05.579034

Biochemical fractionation of human α-Synuclein in a Drosophila model of synucleinopathies

Imomnazarov, K.·Lopez-Scarim, J.·Bagheri, I.·Joers, V.·Tansey, M. G.·Martin-Pena, A.
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Abstract

Synucleinopathies are a group of central nervous system pathologies that are characterized by neuronal accumulation of misfolded and aggregated -synuclein in proteinaceous depositions known as Lewy Bodies (LBs). The transition of -synuclein from its physiological to pathological form has been associated with several post-translational modifications such as phosphorylation and an increasing degree of insolubility, which also correlate with disease progression in post-mortem specimens from human patients. Neuronal expression of -synuclein in model organisms, including Drosophila melanogaster, has been a typical approach employed to study its physiological effects. Biochemical analysis of -synuclein solubility via high-speed ultracentrifugation with buffers of increasing detergent strength offers a potent method for identification of -synuclein biochemical properties and the associated pathology stage. Unfortunately, the development of a robust and reproducible method for evaluation of human -synuclein solubility isolated from Drosophila tissues has remained elusive. Here, we tested different detergents for their ability to solubilize human -synuclein carrying the pathological mutation A53T from brains of aged flies. We also assessed the effect of sonication on solubility of human -synuclein and optimized a protocol to discriminate relative amounts of soluble/insoluble human -synuclein from dopaminergic neurons of the Drosophila brain. Our data established that, using a 5% SDS buffer, the 3-step protocol distinguishes between cytosolic soluble proteins in fraction 1, detergent-soluble proteins in fraction 2 and insoluble proteins in fraction 3. This protocol shows that sonication breaks down -synuclein insoluble complexes from the fly brain, making them soluble in the SDS buffer and enriching fraction 2 of the protocol.

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Imomnazarov, K., Lopez-Scarim, J., Bagheri, I., Joers, V., Tansey, M. G., Martin-Pena, A.. 2024-02-07. Biochemical fractionation of human α-Synuclein in a Drosophila model of synucleinopathies. https://doi.org/10.1101/2024.02.05.579034

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