The Role of SAP97 Isoforms in Regulating Synaptic Function in Hippocampal Neurons

Abstract

SAP97 is a member of the discs-large family of membrane-associated guanylate kinases (MAGUK), and is a multidomain scaffold protein implicated in the forward trafficking and synaptic localization of NMDA- and AMPA-type glutamate receptors. Specifically, SAP97 directly binds the GluR1 subunit of AMPA receptors and NR2 subunits of NMDA receptors and participates in their forward trafficking from the Golgi network to the plasma membrane. Alternative splicing of SAP97 in the N-terminus gives rise to palmitoylated αSAP97 and L27-domain containing βSAP97 isoforms, while insertions between the SH3 and GUK domains results in further isoforms containing the I3, I2, I4 and/or I5 inserts. This study aimed to investigate the effects of SAP97 isoforms on synaptic transmission and plasticity in hippocampal neurons. It is known that the synaptic insertion of GluR1-containing AMPA receptors is critical for synaptic plasticity, under the control of NMDA receptors. However, the mechanisms responsible for GluR1 insertion and retention at the synapse are unclear. Whether SAP97 plays a role in scaffolding GluR1 at the postsynaptic membrane is controversial, attributable to its expression as a collection of alternatively spliced isoforms with ill-defined spatial and temporal distributions. In the present study, we have used electrophysiology and imaging to demonstrate that postsynaptic, N-terminal splice isoforms of SAP97 directly modulate the levels and function of synaptic AMPARs and NMDARs. Overall, αSAP97-I3 and βSAP97-I3/I2 differentially influence the subsynaptic localization and dynamics of AMPARs by creating binding sites for GluR1-containing receptors within synaptic and extrasynaptic subdomains. With regards to synaptic plasticity, i.e. long-term potentiation (LTP) and depression (LTD), we found that both α- and β-isoforms of SAP97-I3 impair LTP but enhance LTD via independent isoform-specific mechanisms. By recording from pairs of synaptically coupled hippocampal neurons, we show that αSAP97-I3 occludes LTP by enhancing the levels of postsynaptic AMPA receptors, while βSAP97 blocks LTP by reducing the synaptic localization of NMDA receptors. Examination of the surface pools of AMPA and NMDA receptors indicates that αSAP97-I3 selectively regulates the synaptic pool of AMPA receptors, whereas βSAP97-I3 and βSAP97-I2 regulate the extrasynaptic pools of both AMPA and NMDA receptors. Knockdown of βSAP97-I3 increases the synaptic localization of both AMPA and NMDA receptors, showing that endogenous βSAP97-I3 restricts glutamate receptor expression at excitatory synapses. This isoform-dependent differential regulation of synaptic versus extrasynaptic pools of glutamate receptors will determine how many receptors are available for the induction and the expression of synaptic plasticity. These results indicate that N-terminal splicing of SAP97 can regulate the ability of synapses to undergo plasticity by controlling the surface distribution of AMPA and NMDA receptors.