THE IMPACT OF Α-SYNUCLEIN ON MICROTUBULES:FROM DYNAMICS TO ULTRASTRUCTURE

α-Synuclein is a presynaptic protein supposed to be involved in the control of neuronal synapse functions. It is widely expressed in brain tissue and associated to Parkinson's disease. When free in the cytoplasm, α-synuclein is unstructured, while it adopts a α-helical conformation when bound to vesicles. Its variable structure allows α-synuclein to interact with multiple partners and makes difficult to understand its physiological role, which remains elusive despite decades of intense study. Here, we looked at the interaction between α-synuclein and microtubules, using both wild type and mutated α-synuclein. We investigated the influence of α-synuclein on microtubule nucleation and dynamics and on microtubule structure. We found that α-synuclein is a novel, foldable, microtubule dynamase, which could participate in the organization of the microtubule cytoskeleton at the pre-synapse, through its binding to tubulin and its regulation of microtubule nucleation and dynamics. We also showed that α-synuclein mutants are much less sensitive than wild type α-synuclein to fold upon tubulin binding and are more prone to cause tubulin aggregation rather than polymerization. Next, we found that α-synuclein deeply affects the structure of microtubules assembled in vitro causing changes in some of the parameters that define it, namely microtubule diameter and tubulin periodicity. Wild type α-synuclein increases the microtubule diameter, but has no effects on tubulin periodicity. A30P α-synuclein, instead, increases both these parameters and A53T and E46K α-synuclein decrease them. We also analysed 3D reconstructions of microtubules and unravelled some very particular structures assembled in the presence of mutated α-synuclein. Next, we carried out an extensive study of the protofilament number distribution among the microtubule population by use of cryo-electron microscopy and we discovered that α-synuclein increases the presence of microtubules with uncommon structures, especially highly twisted microtubules and microtubules with a small number of protofilaments. We also found that the protofilament distribution changes with the time of polymerization. Finally, we showed that the amount of E46K α-synuclein bound to microtubules is significantly higher than that of the other variants. These results support the idea that the interaction of α-synuclein with microtubules heavily impacts on microtubules and, consequently, could play an important role in modulating synaptic physiology. In addition, its alteration can reasonably cause neuronal dysfunction via impairment of the proper microtubule organization and structure. The interaction between α-synuclein and microtubules seems to be very complex, changes over time and depends on the proper folding of α-synuclein. Finally, our results suggest that pathological variants of α-synuclein impair the microtubule system promoting tubulin aggregation more than polymerization, but also changing the structure and the stability of the microtubules that are formed. Thus, this work provides new evidences for looking at the regulation of microtubule as a crucial step in the pathogenesis of Parkinson’s disease.