Levetiracetam influences downstream protein interactions at synapses expressing SV2A

Although levetiracetam is one of the most widely prescribed antiepileptic drugs, its mechanism of action remains unknown beyond the binding to the synaptic vesicle protein SV2A. SV2A is one of the three paralogs of the SV2 family. The function of SV2 has not been fully elucidated yet, however it is known that SV2A is involved in synaptic endocytosis and vesicle protein trafficking; in particular, it regulates vesicular levels of synaptotagmin, the calcium sensor for neurotransmitter release. Here, we tested the hypothesis that levetiracetam affects synaptic vesicle composition. Levetiracetam chronic treatment post-transcriptionally decreased several vesicular proteins and increased LRRK2, without any change in mRNA levels. Analysis of SV2A interactome indicated that the presynaptic proteins regulations induced by levetiracetam are mediated through protein-protein interactions. Next, we asked whether levetiracetam affects the trafficking function and protein-protein interactions of SV2A. To this end, a new approach to isolate newly endocytosed vesicles was developed. Unbiased proteomics results revealed that levetiracetam changes the association of APT1 and complexin-1 (a synaptotagmin interactor) to newly endocytosed vesicles. Protein-protein interaction studies suggested that levetiracetam alters the interaction of synaptotagmin with SV2A and complexin. Importantly, levetiracetam effects predominated in the absence of SV2B, suggesting a possible mechanism of pharmacological specificity. We propose that levetiracetam shapes the mechanism of neurotransmitter release by altering SV2A trafficking function and that SV2 paralogs’ expression pattern underlies pharmacological specificity.