On the Development of a Technique to Isolate Newly Recycled Synaptic Vesicles for the Purpose of Studying the Mechanism of Action of the Anticonvulsant Drug Levetiracetam

Abstract

Levetiracetam (lev) is a clinically used anti-epileptic drug. In contrast to traditional anti-epileptic drugs that target ion channels or GABAergic neurotransmission in order to globally depress synaptic activity, lev appears to have an activity-dependent effect. Lev binds the synaptic vesicle protein SV2 to depress synaptic transmission via an unknown mechanism. It was discovered that the depressive effect of lev on neurotransmission in cultured neurons was only apparent after high frequency stimulus trains. This observation has been interpreted to indicate that vesicle fusion is required for lev to reach the SV2A present in vesicles, but this hypothesis does not account for the fact that lev is equally soluble in chloroform and water and has been demonstrated to be membrane permeable. We propose that vesicles must be recycled in order for lev to act because it changes protein trafficking. Lev’s target, SV2A, has been shown to play a role in protein trafficking during the recycling of synaptic vesicles. SV2 content in synaptic vesicles has been shown to be tightly regulated; exactly 5 copies are present in greater than 95% of vesicles, consistent with it being a potential nucleator of endocytosis. Furthermore, SV2 contains a domain that interacts with multiple clathrin adaptor proteins, and altering expression or a binding site can influence the sorting of synaptotagmin, a synaptic calcium sensor, into vesicles. To test the hypothesis that lev alters protein trafficking in synaptic vesicles via an interaction with SV2A, I aim to studying the composition of recycling synaptic vesicles in the presence and absence of levetiracetam. To do this, I will characterize and vet a protocol under development in the Bajjalieh lab that uses a recently developed class of dynamin inhibitors to trap vesicles in the process of endocytosis. Preliminary data indicates that we are able to isolate synaptosomes; depolarize them in the presence of a dynamin inhibitor, which traps recycling vesicles on the plasma membrane; isolate trapped vesicles via density centrifugation; and release vesicles from the plasma membrane with purified dynamin and GTP. Western blot analysis of these released vesicles suggests that lev changes the stoichiometry of vesicle proteins, supporting an effect on trafficking.