Levetiracetam accelerates the onset of supply rate depression in synaptic vesicle trafficking.

OBJECTIVE: To determine if levetiracetam (LEV) enhances the impact in excitatory presynaptic terminals of a rate-limiting mechanism in vesicle trafficking termed supply rate depression that emerges to limit synaptic transmission during heavy, epileptiform use. METHODS: The effect of LEV was measured with electrophysiologic assays of monosynaptic connections in ex vivo hippocampal slices from wild-type and synapsin knockout mice, and in primary cell culture neurons from wild-type and synaptic vesicle glycoprotein 2a (SV2a) knockout mice. RESULTS: LEV enhanced the impact of supply rate depression at Schaffer collateral synapses by shortening the time course for induction. The LEV effect was selective for supply rate depression because other presynaptic vesicle trafficking mechanisms were not affected. The half maximal effective concentration (EC50 ) was ~50 µm. The maximal effect was ~15% and occurred at 100 µm, which is a clinically relevant concentration. An experimental protocol is established for distinguishing atypical antiepileptic drugs (AEDs) that affect supply rate depression, such as LEV, from typical AEDs, such as carbamazepine, that affect upstream mechanisms. The LEV effect was abolished at synapses from knockout mice lacking SV2a and from synapses lacking synapsin 1 and 2. SIGNIFICANCE: The findings are consistent with the new hypothesis that LEV acts to treat epilepsy by accelerating the induction of supply rate depression at excitatory synapses during incipient epileptic activity. The absence of the effect in the knockouts confirms that presynaptic function is the target. More specifically, the absence in SV2a knockouts is consistent with previous binding studies suggesting that SV2a is the target for LEV. The absence in synapsin knockouts indicates that the phenotypic target intersects with the biochemical pathway that is altered in synapsin knockouts. The results from synapsin knockouts additionally suggest that development of functional analogs with increased potency might be possible because induction of supply rate depression is faster in synapsin knockouts compared to wild-type synapses treated with LEV.

GluN3A promotes dendritic spine pruning and destabilization during postnatal development.

Synaptic rearrangements during critical periods of postnatal brain development rely on the correct formation, strengthening, and elimination of synapses and associated dendritic spines to form functional networks. The correct balance of these processes is thought to be regulated by synapse-specific changes in the subunit composition of NMDA-type glutamate receptors (NMDARs). Among these, the nonconventional NMDAR subunit GluN3A has been suggested to play a role as a molecular brake in synaptic maturation. We tested here this hypothesis using confocal time-lapse imaging in rat hippocampal organotypic slices and assessed the role of GluN3A-containing NMDARs on spine dynamics. We found that overexpressing GluN3A reduced spine density over time, increased spine elimination, and decreased spine stability. The effect of GluN3A overexpression could be further enhanced by using an endocytosis-deficient GluN3A mutant and reproduced by silencing the adaptor protein PACSIN1, which prevents the endocytosis of endogenous GluN3A. Conversely, silencing of GluN3A reduced spine elimination and favored spine stability. Moreover, reexpression of GluN3A in more mature tissue reinstated an increased spine pruning and a low spine stability. Mechanistically, the decreased stability in GluN3A overexpressing neurons could be linked to a failure of plasticity-inducing protocols to selectively stabilize spines and was dependent on the ability of GluN3A to bind the postsynaptic scaffold GIT1. Together, these data provide strong evidence that GluN3A prevents the activity-dependent stabilization of synapses thereby promoting spine pruning, and suggest that GluN3A expression operates as a molecular signal for controlling the extent and timing of synapse maturation.

Tyrosine phosphorylation regulates the endocytosis and surface expression of GluN3A-containing NMDA receptors.

Selective control of receptor trafficking provides a mechanism for remodeling the receptor composition of excitatory synapses, and thus supports synaptic transmission, plasticity, and development. GluN3A (formerly NR3A) is a nonconventional member of the NMDA receptor (NMDAR) subunit family, which endows NMDAR channels with low calcium permeability and reduced magnesium sensitivity compared with NMDARs comprising only GluN1 and GluN2 subunits. Because of these special properties, GluN3A subunits act as a molecular brake to limit the plasticity and maturation of excitatory synapses, pointing toward GluN3A removal as a critical step in the development of neuronal circuitry. However, the molecular signals mediating GluN3A endocytic removal remain unclear. Here we define a novel endocytic motif (YWL), which is located within the cytoplasmic C-terminal tail of GluN3A and mediates its binding to the clathrin adaptor AP2. Alanine mutations within the GluN3A endocytic motif inhibited clathrin-dependent internalization and led to accumulation of GluN3A-containing NMDARs at the cell surface, whereas mimicking phosphorylation of the tyrosine residue promoted internalization and reduced cell-surface expression as shown by immunocytochemical and electrophysiological approaches in recombinant systems and rat neurons in primary culture. We further demonstrate that the tyrosine residue is phosphorylated by Src family kinases, and that Src-activation limits surface GluN3A expression in neurons. Together, our results identify a new molecular signal for GluN3A internalization that couples the functional surface expression of GluN3A-containing receptors to the phosphorylation state of GluN3A subunits, and provides a molecular framework for the regulation of NMDAR subunit composition with implications for synaptic plasticity and neurodevelopment.