Elucidating Levetiracetam action at the presynaptic terminal
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Ciruelas, Kristine
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Levetiracetam (KeppraTM, LEV) is the first of a growing class of anti-epileptic drugs that shows great promise in the treatment of multiple neurological disorders. LEV has a unique receptor, Synaptic Vesicle Protein 2A (SV2A), which is both necessary and sufficient for binding. SV2A is the most widely expressed member of a three-protein family (SV2A, B, C) that is localized exclusively to vesicles that undergo calcium-stimulated secretion. Although multiple functions have been ascribed to SV2, it has one verified function. All three SV2s bind to and regulate the stability and trafficking of synaptotagmin, the calcium sensing protein that mediates fast, evoked transmitter release. Although LEV decreases synaptic transmission in ex vivo preparations, it has failed to show effects on isolated, autaptic (self-synapsing) neurons, complicating elucidation of its molecular mechanism of action. We asked if the absence of a LEV effect in autaptic neurons was due to the presence of non-LEV binding SV2 paralogs. While LEV had no effect on synaptic transmission in neurons from wild-type (WT) mice that express both SV2A and SV2B, it decreased synaptic depression in neurons cultured from SV2B knockout mice (BKO). This suggests that LEV inhibits SV2 function at synapses that express primarily or exclusively SV2A. To test hypotheses of LEV’s molecular action, we first focused on SV2’s well-established role in regulating the stability and trafficking of synaptotagmin. LEV disrupted the interaction between SV2A and synaptotagmin, resulting in decreased and slowed internalization of synaptotagmin in neurons from BKO but not WT mice. To determine if LEV impacted the trafficking of other proteins, we developed a technique to isolate newly recycled vesicles from synaptic terminals (synaptosomes). Proteomic analyses of this preparation revealed a LEV-dependent decrease in the amount of complexin1, a soluble protein that binds synaptotagmin and the fusion protein (SNARE) complex in vesicles isolated from BKO but not WT mice. This suggests LEV affects SV2A’s protein-protein interactions and that further LEV studies should focus on synapses solely expressing SV2A. Two classes of neurons fit this category, most inhibitory neurons and the excitatory granule cells of the dentate gyrus. These two classes have also been implicated in epilepsy.
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