Discovering the Mechanism Regulating EphB/ephrin-B Dependent Interaction at Synapses: with an Emphasis on NMDAR-EphB Interaction and Ephrin-B3-PSD-95 Interaction
Synapses are transcellular structures that form between neurons. They are highly complex, due to the large number of neurons to which they connect and to the variety proteins present at synapses. Hundreds to thousands of proteins can be found at mammalian synapses, enabling complicated protein interactions both in trans and in cis at synapses. The goal of my thesis is to investigate how particular protein interactions that play key roles in the development and function of synapses are regulated. A better understanding of the molecular basis and function of these interactions will advance our understanding of how the complex brain function is built upon those fundamental protein interactions. EphBs and ephrin-Bs are pairs of synaptic adhesion molecules that can be localized either pre- or post-synaptically. EphB/ephrin-B can modulate synapse function through interacting with other molecules, such as the N-methyl-D- aspartate receptor (NMDAR), a subtype of glutamate receptors that plays a central role in neuronal development, plasticity, neural function, and neurotoxicity. At excitatory synapses, EphBs interact with NMDAR and regulate NMDAR synaptic localization and function. The NMDAR-EphB interaction is known to depend on the extracellular domain of both proteins, but the specific amino acids required for the interaction remain unknown. Here I show that: 1) EphBs are phosphorylated extracellularly upon ephrinB activation. Phosphorylation of a specific extracellular tyrosine residue located at the fibronectin type III (FN3) region is required for the interaction with NMDAR; and 2) The GluN1 subunit N terminal domain (NTD) hinge region is required for interaction with EphB. I demonstrate that the positive charge of the exposed surface and glycosylation of this region are required for the EphB-NMDAR interaction. Moreover, I show disruption of the NMDAR-EphB interaction increases NMDAR mobility. Consistent with previous work showing the importance of the EphB-NMDAR interaction in mature neurons, the mobility of NMDAR is regulated developmentally with EphB-dependent regulation of NMDAR retention occurring only in mature neurons. Besides NMDAR, EphB/eprhin-B are also involved in interacting with another molecule: PSD-95. PSD-95 is an important scaffolding protein enriched at post synaptic density. However, it is unclear how it is localized to synaptic sites. I contributed to our recent study that showed that ephrin-B3 controls the synaptic localization and stability of PSD-95 through direct interaction (Hruska et al., 2015) . My work on this project demonstrated that knocking down PSD-95 does not affect the synaptic localization and expression pattern of ephrin-B3. This was a key finding that enabled us to support the model that ephrinB3 functions upstream of PSD-95 to retain PSD-95 at synaptic sites. Overall, my findings define three novel mechanisms for EphB and ephrin-B dependent interactions with important excitatory synaptic proteins: NMDAR and PSD95, respectively. Importantly my data provide evidence for how synaptic localization and retention of the NMDAR and PSD-95 occur. These findings will likely have a broad impact, both for the understanding of synaptic function and for understanding what goes wrong at synapses during diseases, particularly diseases where the proper localization of synaptic proteins is defective.
Xia, Nan, "Discovering the Mechanism Regulating EphB/ephrin-B Dependent Interaction at Synapses: with an Emphasis on NMDAR-EphB Interaction and Ephrin-B3-PSD-95 Interaction" (2018). ETD Collection for Thomas Jefferson University. AAI10747650.