The properly regulated secretion of neurotransmitter in the brain is essential for normal functioning of the brain. Normal neuronal excitability is disrupted in epilepsy. Hyper-excitable neurons spontaneously fire, become synchronized, and start a wave of neuronal firing which can cause a seizure. Levetiracetam (KeppraTM (UCB)) is an antiepileptic drug that is used with great success to treat certain forms of epilepsy. Synaptic vesicle protein 2A (SV2A), an integral synaptic vesicle membrane protein, was recently identified as the target of levetiracetam. This makes SV2A the first example of a presynaptic protein as a target for antiepileptics. SV2 proteins are essential for the proper release of neurotransmitters and SV2A knockout mice develop severe seizures. We propose a model for the mechanism of action of levetiracetam and we will test this model by using a combination of molecular biological, biochemical and pharmacological approaches:
Aim1. Map the phosphorylation sites of human SV2A: SV2A protein will be purified from residual human brain tissue collected during surgery from epileptic patients using a specific SV2A antibody. The phosphorylation sites in human SV2A protein will then be determined by mass spectroscopic analysis.
Aim2. Generate specific antibodies for phospho-SV2A: Phospho-peptides corresponding to the characterized phosphorylation sites in human SV2A will be used to generate specific monoclonal antibodies. An ELISA assay will be developed to sensitively measure the level of SV2A phosphorylation in human tissue.
Aim3. Investigate changes in the phosphorylation state of human SV2A during epilepsy: The phosphorylation state of SV2A in residual tissue collected during surgery from epileptic patients and in control tissue will be determined using the developed ELISA assays. This experiment will test the hypothesis that epilepsy in human tissue leads to changes in the phosphorylation of SV2A and if levetiracetam treatment affects these changes.
Epilepsy is a neurological disease that affects about 1% of the population. We will study the role of a protein called SV2A that has been recently identified as drug target for antiepileptic drugs. We anticipate that our studies will help in the better understanding of the molecular processes that cause epilepsy and in the development of new treatments for epilepsy.
