Previous studies have revealed a role for EphrinB/EphB signaling in the development of excitatory synapses (Klein, 2009
). However, the regulatory constraints that temper EphB-dependent synapse development so that excitatory synapses form at the right time and place, and in the correct number were not known. In this study we identify a RhoA GEF, E5, which functions to restrict EphB-dependent excitatory synapse development. E5 interacts with EphB prior to EphrinB binding, and by activating RhoA serves to inhibit synapse development. The binding of EphrinB to EphB as synapses form triggers the phosphorylation and degradation of E5 by a Ube3A-dependent mechanism. The reduction in E5 expression may allow EphB to promote excitatory synapse development by activating Rac and other proteins at the synapse.
The findings that E5 functions to restrict excitatory synapse number suggests that, even though EphBs promote excitatory synapse development, there are constraints on the activity of EphB so that synapse number is effectively controlled. There are several steps in the process of synapse development where E5 may function to restrict synapse number. One possibility is that E5 functions early in development as a barrier to excitatory synapse formation by activating RhoA and restricting the motility or growth of dendritic filopodia that are the sites of contact by the presynaptic neuron. For example, by inhibiting dendritic filopodia formation or motility, E5 may decrease the number of contacts the filopodia make with the presynaptic neuron, thus resulting in the formation of fewer synapses. An alternative possibility is that E5 functions to restrict synapse number later in development perhaps to counterbalance the positive effects of EphB on Rac that promote dendritic spine development. An additional possibility is that E5 functions after excitatory synapse development as a regulator of synapse elimination.
Our analyses of E5 function are most consistent with the possibility that E5 functions early in the process of synapse development. First, we find that E5 is expressed, active, and bound to EphB prior to synapse formation. Second, the interaction of EphrinB with EphB, a process that is thought to be an early step in excitatory synapse development, triggers the degradation of E5. Third, our preliminary time-lapse imaging studies suggest that E5 is localized to newly formed filopodia prior to synapse development where it appears to restrict filopodia motility and growth (Margolis et al. unpublished). Thus, E5 might function as an initial barrier to synapse formation until it is degraded upon EphrinB binding to EphB.
It is possible that through its interaction with EphB, E5 marks the sites where synapses will form, and that the degradation of E5 is a critical early step in excitatory synapse development. While the mechanisms by which E5 is degraded are not fully understood, our studies suggest that the phosphorylation of the N-terminus of E5 at Y361 triggers the Ube3A-mediated proteasomal degradation of E5. One possibility is that prior to pY361 the N- and C-terminal portions of E5 interact, thereby protecting E5 from degradation. The phosphorylation of E5 at Y361 may relieve this inhibitory constraint allowing for E5 ubiquitination and degradation. A similar mechanism has been shown to regulate the activation of the Rac GEF Vav, (Aghazadeh et al., 2000
)). During EphrinA/EphA signaling it has been proposed that Vav-mediated endocytosis of the EphrinA/EphA complex may allow the conversion of the initial adhesive interaction between EphrinA and EphA-expressing cells into a repulsive interaction that results in growth cone collapse and axon repulsion. It is possible that E5 has a related function during EphB signaling at synapses. Typically the EB/EphB interaction is thought to be repulsive. This has been documented in studies of EphB’s role in the process of axon guidance (Egea and Klein, 2007
; Flanagan and Vanderhaeghen, 1998
). However, during synapse development the EphrinB/EphB interaction is thought to result in synapse formation, a process that requires an interaction between the developing pre- and post-synaptic specialization. One possibility is that when EphrinB and EphB mediate the interaction between the incoming axon and the developing dendrite, the interaction is facilitated by the degradation of E5 by Ube3A. Since E5 is a RhoA GEF, its presence might initially lead to repulsion between the incoming axon and the dendrite. However, the EphB-dependent degradation of E5 might convert this initial repulsive interaction into an attractive one.
The finding that Ube3A is the ubiquitin ligase that controls EphB-mediated E5 degradation is of interest given the role of Ube3A in human cognitive disorders such as Angelman syndrome and autism. The absence of Ube3A function in Angelman syndrome would be predicted to result in an increase in E5 protein expression, and thus a decrease in EphB-dependent synapse formation. Consistent with this possibility, we find in a mouse model for Angelman syndrome that the level of E5 protein expression is elevated and that in response to EphrinB treatment E5 is not degraded. Likewise, several studies have indicated that synapse development and function is disrupted in these mice (Jiang et al., 1998
; Yashiro et al., 2009
The recent finding that the Ube3A gene lies within a region of chromosome 15 that is sometimes duplicated in autism raises the possibility that altered levels of Ephexin5 and the resulting defects in excitatory synapse restriction might also be a mechanism relevant to the etiology of autism (Glessner et al., 2009). If this is the case, a possible therapy for treating autism might be to reduce the level of Ube3A activity, and thus increase the level of Ephexin5 expression. It is important to consider that in addition to Ephexin5, Ube3A regulates the abundance of other synaptic proteins. Nevertheless, the ultimate effect of the aberrant expression of Ephexin5 and other Ube3A substrates on synapse development and function will require further study. It seems likely that such studies will provide further understanding of the development of human cognitive function and new insights into how this process goes awry in disorders such as Angelman syndrome and autism.