Investigating the role of a-Synuclein in presynaptic microtubule dynamics in dopaminergic neurons

a-synuclein, a protein involved in Parkinson's Disease, was previously shown to be physiologically implicated in neurotransmitter release through an ill-defined mechanism. In disease states, a-synuclein undergoes a conformational change from a partial a-helix structure to a pathological prone-to-aggregate ß-sheet conformation. This switch leads a-synuclein to lose its function and to aggregate into toxic oligomers and fibrillar structures, which ultimately can cause the death of the dopaminergic neurons of the substantia nigra pars compacta. Microtubules are dynamic polymers composed of regulated a- and ß- tubulin subunit assembly. In neurons they play an essential role by maintaining the cytoarchitecture and serving as tracks for motor proteins, thus regulating both axonal transport and synaptic activity. Recent findings indicate that microtubules interact with different Parkinson's Disease related proteins, including a-synuclein. In particular, a-synuclein and a-tubulin directly associate in the healthy human brain, and that this interaction is mostly localized in the presynaptic compartment. The aim of my project is to determine whether a-synuclein regulates microtubule dynamics at presynaptic contacts and, if so, whether mutations in a-synuclein associated to Parkinson disease can affect this activity. To this end, I will adopt dopaminergic neurons isolated from ventral midbrain and cultivated in vitro to follow microtubule dynamics by transiently expressing fluorescently tagged EB3, a neuronal microtubule-associated protein that marks the plus ends of growing microtubules together with presynaptic markers. The role of a-synuclein in synaptic microtubule dynamic behaviour will be investigated by carrying out these microscopy assays in the absence of a-synuclein expression or in the presence of the most common Parkinson's disease-related mutated forms of a-synuclein to evaluate the role of this putative microtubule activity in Parkinson's disease. By identifying a-synuclein as a regulator of synaptic microtubule dynamics, these experiments promise to shed light on the physiological relevance of the newly identified interaction between a-synuclein and microtubules at presynaptic contacts, and generate mechanistic insights into the putative role of a-synuclein in neurotransmitter release.