Trans-synaptic EphB2–ephrin–B3 interaction regulates excitatory synapse density by inhibition of postsynaptic MAPK signaling

Abstract

Nervous system function requires tight control over the number of synapses individual neurons receive, but the underlying cellular and molecular mechanisms that regulate synapse number remain obscure. Here we present evidence that a trans-synaptic interaction between EphB2 in the presynaptic compartment and ephrin-B3 in the postsynaptic compartment regulates synapse density and the formation of dendritic spines. Observations in cultured cortical neurons demonstrate that synapse density scales with ephrin-B3 expression level and is controlled by ephrin-B3-dependent competitive cell-cell interactions. RNA interference and biochemical experiments support the model that ephrin-B3 regulates synapse density by directly binding to Erk1/2 to inhibit postsynaptic Ras/mitogen-activated protein kinase signaling. Together these findings define a mechanism that contributes to synapse maturation and controls the number of excitatory synaptic inputs received by individual neurons.N euronal activity is a key determinant of maintaining and controlling the number of synaptic connections (1, 2), but the molecular mechanisms likely to establish the normal density of synaptic contacts are still not well defined (3). Synapse density could be controlled by either secreted or cell surface molecules, but trans-cellular interactions are attractive because of their ability to coordinate events between cells. One family of synaptic adhesion molecules that is well suited to control synapse density is ephrin-Bs (eBs), a family of three (eB1-3) ligands for the EphB family of receptor tyrosine kinases (4). Recent work suggests that ephrins may negatively regulate Ras/MAPK activation in nonvertebrate systems during morphogenesis (5), and that MAPK signaling can negatively regulate presynaptic terminal maturation (6, 7). Here we show that eB3 controls synapse density and the formation of dendritic spines through a competitive postsynaptic mechanism relying on inhibition of MAPK signaling. ResultsEphrin-B3 Expression Level Controls Synapse Density. Early in development [5-10 days in vitro (DIV 5-10)], there are few dendritic spines and synapses that are rapidly added on the dendritic shaft. During this time, eB3 is enriched at excitatory synapses but is also localized to many extrasynaptic locations (Fig. 1A). As neurons mature (DIV 14-21), dendritic spines are formed and become the principal site of excitatory synapses (8); eB3 becomes largely restricted to spine and shaft excitatory synaptic contacts (Fig. 1A and Fig. S1A). The synaptic pattern of eB3 expression suggests that it may be involved in the formation and maturation of excitatory synapses. We asked whether the expression level of eB3 is related to the density of excitatory synapses on individual neurons by plotting the density of endogenous PSD-95 puncta vs. two different measures of eB3 expression: the density of eB3 puncta and the overall intensity of eB3 immunofluorescence. We found that both measures of eB3 expression vary from neuron to neuron and are correlated with PSD-95 punct...

Rheb activation disrupts spine synapse formation through accumulation of syntenin in tuberous sclerosis complex

“…The reason for this difference is unclear, but our overexpression of ephrinB3 plasmid was longer and stronger (10 days under the control of the CAG promoter) than that in the other study 32 (4 days under the control of the cytomegalovirus promoter). Because ephrinB3 regulates synapse density by inhibiting the postsynaptic mitogenactivated protein kinase signalling 31 that is required for increases in spine density 33 , long and strong expression of ephrinB3 might reduce spine synapse density. Future studies will be needed to characterize this mechanism.…”

“…Syntenin has been demonstrated to be a downstream effector of ephrinB3 (ref. 30), which modulates glutamatergic synaptic density 31 , particularly on shaft synapses 32 . To test whether syntenin is involved in excitatory shaft synapse formation, we knocked down syntenin in WT and Tsc2 þ / À neurons.…”

Eph receptors and ephrins in neuron–astrocyte communication at synapses

“…Interestingly, at mature rodent CA3-CA1 synapses, both EphB receptors and ephrin-Bs are postsynaptically localized (Bouvier et al, 2008;Buchert et al, 1999;Grunwald et al, 2004;Henkemeyer et al, 2003;McClelland et al, 2009;Torres et al, 1998). Although the degree to which EphB receptors and ephrin-Bs colocalize with each other at synaptic sites remains to be fully explored, several studies point to multiple functions of postsynaptic ephrin-Bs in spine morphogenesis (McClelland et al, 2010;Segura et al, 2007), glutamatergic synapse formation on dendritic shafts (Aoto et al, 2007), constitutive regulation of glutamate receptor turnover (Essmann et al, 2008) and synaptic plasticity (Bouzioukh et al, 2007), and more recently in cis-attenuation of postsynaptic EphB function and signaling (Antion et al, 2010). Thus, complementary and overlapping distributions of EphB receptors and ephrin-Bs at synaptic sites contribute to the complex mechanisms through which these Eph/ephrin family members regulate synapses in the brain.…”

Slippery signaling: Palmitoylation-dependent control of neuronal kinase localization and activity

“…Signaling via Ephs and their ephrin ligands regulates a diverse array of processes, including axon guidance, synaptogenesis and the stability and plasticity of mature synapses (Kania and Klein, 2016, McClelland et al, 2010, Klein, 2009). It will be interesting to determine the extent to which the diverse roles of ephrin/Eph signaling in neuronal development and function are palmitoylation-dependent.…”