Intracellular and Trans-Synaptic Regulation of Glutamatergic Synaptogenesis by EphB Receptors

Abstract

The majority of mature excitatory synapses in the CNS are found on dendritic spines and contain AMPA-and NMDA-type glutamate receptors apposed to presynaptic specializations. EphB receptor tyrosine kinase signaling has been implicated in both NMDA-type glutamate receptor clustering and dendritic spine formation, but it remains unclear whether EphB plays a broader role in presynaptic and postsynaptic development. Here, we find that EphB2 is involved in organizing excitatory synapses through the independent activities of particular EphB2 protein domains. We demonstrate that EphB2 controls AMPA-type glutamate receptor localization through PDZ (postsynaptic density-95/Discs large/zona occludens-1) binding domain interactions and triggers presynaptic differentiation via its ephrin binding domain. Knockdown of EphB2 in dissociated neurons results in decreased functional synaptic inputs, spines, and presynaptic specializations. Mice lacking EphB1-EphB3 have reduced numbers of synapses, and defects are rescued with postnatal reexpression of EphB2 in single neurons in brain slice. These results demonstrate that EphB2 acts to control the organization of specific classes of mature glutamatergic synapses.

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

“…3D). To address whether cortical neurons from eB3 −/− mice have fewer synapses, we determined synapse density in layer 5 of cortical cryosections from wt and eB3 −/− mice by measuring overlap of pre-and postsynaptic markers (10). Consistent with previous reports (11-13), we found no significant difference in excitatory synaptic density between wt and eB3 −/− mice (Fig.…”

“…Primary dissociated rat cortical neurons and transfections were conducted as described previously (10,15). Primary dissociated cortical neurons from eB3 −/− mice and wt littermate controls were prepared from postnatal day 1 (P1) mice.…”

“…Primary dissociated cortical neurons from eB3 −/− mice and wt littermate controls were prepared from postnatal day 1 (P1) mice. Organotypic slice cultures were prepared and transfected as previously described (10).…”

“…Cells and tissue were fixed and stained as described previously (10,15). Antibodies used were: chicken anti-GFP (1:2,500; Upstate), rabbit anti-Erk1/2 (1:500; Promega), rabbit anti-eB3 (1:50; specificity confirmed by observing loss of staining after incubation with acetone-precipitated proteins from wt but not eB3 −/− brain tissue; Zymed), mouse anti-PSD-95 (1:250; Affinity BioReagents), rabbit anti-SynGAP (1:1,000; Affinity BioReagents), and guinea pig anti-VGlut1 (1:5,000; Chemicon).…”

Cell–cell signaling via Eph receptors and ephrins

“…Interestingly, based on the known Eph/ephrin complex structure and assuming that the other extracellular regions adopt relatively extended conformations, the distance between the membranes of two interacting cells can be estimated at ~20 nm -consistent with the size of the synaptic cleft. In a recent extension of these studies Dalva and colleagues show that B-class ligand/receptor binding induces a direct interaction at the synapses between EphB and NMDA-type glutamate receptors involving only the extracellular regions of the molecules and independent of the receptor kinase activity [37]. These results confirm that EphB activation promotes Eph/NMDA-R assemblies that are essential for synapse development or function.…”