Roles for alpha-Synuclein in Clathrin-Mediated Synaptic Vesicle Recycling - Project Summary
-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.