The Role of αSAP97 and βSAP97 at the Glutamatergic Synapse

Abstract

Aim The location and density of synaptic AMPA receptors is controlled by scaffolding proteins that lie directly beneath the postsynaptic membrane. Two N-terminal isoforms of SAP97, αSAP97 and βSAP97, are thought to play opposite and complimentary roles in regulating surface AMPA receptor location. Electrophysiology evidence suggests that αSAP97 increases synaptic strength by clustering AMPA receptors at synapses, whereas βSAP97 decreases synaptic strength by scaffolding these receptors at perisynaptic locations. However, this hypothesis has not yet been verified with imaging, as the neuronal synapse lies below the resolution limit of conventional optical microscopy. Methods Cultured rat hippocampal neurons were transiently transfected with α or βSAP97-eGFP, short-hairpin RNA to knock-down βSAP97 expression, or an empty eGFP control plasmid. A single molecule localisation method of super resolution imaging known as dSTORM was used to image the distribution of surface AMPA receptors on transfected neurons. Confocal microscopy was used to quantify both the size of these neurons, and the morphology and density of their synapses. Results Both α and βSAP97 overexpression enhanced the clustering of GluA1-containing AMPA receptors at synapses compared to eGFP controls, whereas acute knock-down of βSAP97 relocated the receptors to perisynaptic sites (p < 0.001). In addition, overexpression of βSAP97 increased the density of GluA1 subunits at synapses (p < 0.001), and reduced the proportion of mature, mushroom-shaped dendritic spines (p = 0.029). Both α and βSAP97 overexpression increased the size of the presynaptic active zone via transsynaptic signalling (αSAP97: p < 0.001; βSAP97: p = 0.024). Compared to αSAP97, βSAP97 overexpression reduced the size of the dendritic tree (p = 0.027), and the proportion of postsynaptic densities with direct access to the presynaptic active zone (p = 0.003). Conclusions The previously observed differences in synaptic strength between α and βSAP97 expressing neurons can be explained by differences in the number of synapses, rather than the location of surface AMPA receptors on the postsynaptic membrane. Both α and βSAP97 can scaffold AMPA receptors at synapses, whereas βSAP97 can actively promote synaptic incorporation of AMPA receptors by releasing them from the perisynaptic pool.