?-Synuclein is a small protein mutated and aggregated in a variety of neurodegenerative diseases. Its physiological function has recently been discovered as a regulator of clathrin mediated synaptic vesicle endocytosis. Nevertheless, the mechanism controlling this event remains unknown. In the current research proposal, we hypothesize that in physiological conditions, ?-synuclein mediates the recruitment of the clathrin adaptor AP2 to the synaptic membrane, thereby facilitating clathrin mediated endocytosis. This hypothesis is based on strong preliminary data showing a significant and selective decrease in the recruitment of AP2 to synaptic membranes when ?-synuclein is acutely depleted from brain cytosol.
In Aim 1, we propose to characterize the ?-synuclein dependent AP2 recruitment to synaptic membrane. Additionally, we will determine the protein interactions undelaying the ?-synuclein/AP2 functional link by performing Aim 2. To characterize the ?-synuclein dependent AP2 membrane recruitment, we will use membrane recruitment assays. We will test the mutual dependency of AP2 and ?-synuclein on their synaptic membrane interaction by depleting AP2 from brain cytosol and detecting membrane-bond ?-synuclein. We will also test the AP2 recruitment to the membrane in different conditions. This will be achieved by the supplementation of ?-synuclein immunodepleted cytosol with different PD linked ?-synuclein mutants carrying folding and lipid binding modifications. Thus, evaluating the importance of ?-synuclein features on AP2 membrane recruitment. Additionally, main endocytic proteins will be immunodepleted from brain cytosol to test other regulators proteins on AP2 recruitment to synaptic membranes. Lamprey synapse have many advantages including the ability to acutely perturb the presynaptic compartment contents due to the size of the axons. Thus, we will evaluate changes in AP2 localization after different perturbations of the synapse by microinjection of the PD linked ?-synuclein mutants on the lamprey synapse. To determine the protein interactions undelaying the ?-synuclein/AP2 functional link, we will use protein-protein in vitro assays to determine if this interaction is direct or indirect. First, we will make a complete biochemical direct interaction study between ?-synuclein and AP2, with identification of a minimal interaction domain; later we will test the interaction of ?-synuclein with well-known AP2 interactors. To discover unknown AP2/?-synuclein interactors, we will perform an unbiased mass spectrometry determination of common interactors between ?- synuclein and AP2. Lastly, inhibitor peptides of the interaction between AP2 and ?-synuclein will be identify and microinjected in the lamprey synapse to evaluate AP2 localization on synaptic vesicle microdomains. At the completion of this proposal, we will find a mechanism for ?-synuclein dependent AP2 membrane recruitment in physiological conditions, which will show the role for ?-synuclein both in the synapse and the brain. Furthermore, the model generated by this research will predict the pathophysiology of synucleopathies for future research.
?-Synuclein is a protein mutated and aggregated in different neurodegenerative diseases and the mechanism for its physiological function in the neuron is still unknown. We will explore the molecular mechanism for ?- synuclein participation in synaptic vesicle endocytosis by characterizing the functional link between ?-synuclein and AP2, the main clathrin adaptor to synaptic membranes. This research will help us to understand the physiological function of ?-synuclein in neurons.
