Tracking the interaction between α-Synuclein mutants and Tubulin Using DOSY (Diffusion Ordered Spectroscopy) NMR

Microtubule abnormalities in both structure and function are implicated in various neurodegenerative diseases, including Parkinson’s disease (PD), due to their critical role in neuronal microtubule dynamics.1 α-Synuclein is a small protein (~14kDa) highly prone to aggregate, forming toxic oligomers and insoluble fibrils that contribute to PD disease-specific inclusions.2 Interestingly, tubulin initiates and promotes the formation of these α-Synuclein fibrils under physiological conditions in vitro3. Nevertheless, the underlying protein-protein interactions remain poorly characterized and require further investigation to elucidate their mechanisms and implications. To gain deeper insights into the interactions between α-synuclein and tubulin, we employed Nuclear Magnetic Resonance (NMR) analysis and in particular Diffusion Ordered Spectroscopy (DOSY) method. This technique separates molecules based on their diffusion coefficients, which inversely correlate with molecular size: smaller molecules diffuse faster and have higher coefficients, while larger aggregates diffuse more slowly.4 This approach enabled us to monitor changes in α-synuclein upon tubulin addition and the formation of higher-order aggregates. The study included wild-type α-synuclein and PD-linked missense mutations in the α-Synuclein gene (SNCA), such as A30P, E46K, and A53T, associated with familial early-onset PD. 5 Our results confirmed a direct interaction between tubulin and wild-type α-synuclein, indicating a weak yet meaningful interaction. Furthermore, we observed distinct binding affinities between tubulin and PD-associated α-synuclein variants, suggesting variant-specific interactions. Further investigation of α-synuclein aggregation over time revealed that the diffusion coefficient of wild-type α-synuclein remained stable in the presence of Synu Clean-D, a compound known to inhibit synuclein aggregation.6 These findings underline the critical role of tubulin in facilitating α-synuclein aggregation and highlight the differential behaviour of PD-linked variants, providing valuable insights into the molecular mechanisms underlying Parkinson’s disease.