SUMMARY Scaffolding molecules at the postsynaptic membrane form the foundation of excitatory synaptic transmission by establishing the architecture of the postsynaptic density (PSD), but the small size of the synapse has precluded measurement of PSD organization in live cells. We measured the internal structure of the PSD in live neurons at approximately 25 nm resolution using photoactivated localization microscopy (PALM). We found that four major PSD scaffold molecules are each organized in distinctive nanodomains ~80 nm in diameter, intrasynaptic protein ensembles that undergo striking changes over time. Further, the dense subdomains of PSD-95 were preferentially enriched in AMPA receptors more than NMDA receptors. Chronic suppression of activity triggered changes in PSD interior architecture that may help amplify synaptic plasticity. The observed clustered architecture of the PSD controlled the amplitude and variance of simulated postsynaptic currents, suggesting several ways in which PSD interior organization may regulate the strength and plasticity of neurotransmission.

Mechanisms regulating lateral diffusion and positioning of glutamate receptors within the postsynaptic density (PSD) determine excitatory synaptic strength. Scaffold proteins in the PSD are abundant receptor binding partners, yet electron microscopy suggests that the PSD is highly crowded, potentially restricting the diffusion of receptors regardless of binding. However, the contribution of macromolecular crowding to receptor retention remains poorly understood. We combined experimental and computational approaches to test the effect of synaptic crowding on receptor movement and positioning in Sprague Dawley rat hippocampal neurons. We modeled AMPA receptor diffusion insynapseswherethedistributionofscaffoldproteinswasdeterminedfromphotoactivatedlocalizationmicroscopyexperiments,andreceptorscaffold association and dissociation rates were adjusted to fit single-molecule tracking and fluorescence recovery measurements. Simulations predicted that variation of receptor size strongly influences the fractional synaptic area the receptor may traverse, and the proportion that may exchange in and out of the synapse. To test the model experimentally, we designed a set of novel transmembrane (TM) probes. A single-pass TM protein with one PDZ binding motif concentrated in the synapse as do AMPARs yet was more mobile there than the much larger AMPAR. Furthermore, either the single binding motif or an increase in cytoplasmic bulk through addition of a single GFP slowed synaptic movement of a small TM protein. These results suggest that both crowding and binding limit escape of AMPARs from the synapse. Moreover, tight protein packing within the PSD may modulate the synaptic dwell time of many TM proteins important for synaptic function.

Amyotrophic lateral sclerosis (ALS) is a common adult-onset nervous system degenerative disease, characterized by the progressive loss of upper and lower motor neurons. TDP-43 pathology in motor neurons is a hallmark of ALS. In addition, the reduced expression of an RNA-editing enzyme, adenosine deaminase acting on RNA 2 (ADAR2), increases the expression of GluA2 at an unedited glutamine/arginine (Q/R) site in the motor neurons of patients with sporadic ALS. The change in the amino acid residue at the Q/R site of GluR2 results in marked alterations in channel properties of AMPA receptors, which increases Ca²⁺ permeability, and this increase in Ca²⁺ influx plays a key role in the death of motor neurons. ADAR2 mRNA is a target RNA for TDP-43, and TDP-43 plays a regulatory role in the expression of ADAR2. Recently, researchers have explored the possibility of gene therapy for ALS by upregulating ADAR2 in mouse motor neurons using an adeno-associated virus serotype 9 (AAV9) vector that enables gene delivery to a wide array of central neurons after peripheral administration and observed that the expression of exogenous ADAR2 in the central neurons effectively prevented progressive motor dysfunction. AAV9-ADAR2 rescued the motor neurons from death by normalizing TDP-43 expression. Therefore, this AAV9-mediated ADAR2 gene delivery may enable the development of a gene therapy for ALS. i 2014 S. Karger AG, Basel