Shank3 interactions with GKAP and actin help maintain the PSD nanoscale organization

Abstract

Synaptic strength is determined by the number of postsynaptic AMPA-type glutamate receptors (AMPARs) that is activated after pre-synaptic neurotransmitter release. This is dependent on the number of AMPARs that is present in the PSD and their positioning relative to the neurotransmitter release site. PSD95 is a scaffolding protein that is involved in recruiting and placing the AMPARs in the PSD. It is organized into nanodomains that increase in number and density upon induction of LTP. These reorganizations were shown to be dependent on the presence of Shank3. Shank3 is a scaffolding protein that interacts with many other PSD scaffolding molecules and links them to the actin cytoskeleton. Elucidating which of the many possible Shank3 interactions underly the reorganization of PSD95 is essential to increase our understanding of synaptic strength and plasticity. Therefore, we used direct Stochastic Optical Reconstruction Microscopy (dSTORM) imaging of endogenously labelled PSD95, combined with re-expression of specific mutated Shank3 variants to determine which Shank3 interactions influence the PSD95 nanoscale distribution. It was found that weakening the connection with GKAP, the protein that links Shank3 to PSD95, leads to an LTP-like reorganization of PSD95. Additionally, the effects of disrupting Shank3/actin mutations were determined, since Shank3 is perfectly positioned to transduce any changes in the dynamic actin cytoskeleton to the upper PSD. Surprisingly, three mutations that disrupt the interaction between Shank3 and F-actin or actin-binding proteins did not influence PSD95 nanoscale organization. Nevertheless, the actin cytoskeleton does play a role in regulating PSD95 nanoscale organization. Treatment and washout of actin depolymerizing drug Latrunculin B (latB) in rat hippocampal neurons led to an increase in the number of PSD95 nanodomains. Additionally, treatment and wash-out of both LatB and Jasplakinolide (Jasp) impaired LTP-induced PSD95 reorganization. Based on these results, it can be hypothesized that the actin cytoskeleton is able to influence the organization of the upper PSD through Shank3. Regulation by the actin cytoskeleton could stabilize and maintain the PSD95 nanoscale distribution. Release of the control of actin on the PSD, either by decoupling Shank3 from PSD95 or by depolymerizing the actin network, allows PSD95 to reorganize.