Abstract
Little is known about the changes in protein interactions inside synapses during synaptic remodeling, as their live monitoring in spines has been limited. We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction. NMDAR stimulation caused a transient decrease in FRET between the NMDAR and PSD95 in spines of young and mature neurons. The activity of both CaMKII and calpain were essential for this effect in both developmental stages. Meanwhile, inhibition of Src family kinase (SFK) had opposing impacts on this decrease in FRET in young versus mature neurons. Our data suggest concerted roles for CaMKII, SFK and calpain activity in regulating activity-dependent separation of PSD95 from GluN2A or GluN2B. Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.
Highlights
The dynamic molecular reorganization that takes place in dendritic spines is under increasing investigation, as it supports synaptic remodeling, a process required for memory formation
To study the interaction between the NMDA receptor (NMDAR) and PSD95 with Forster Resonance Energy Transfer (FRET)-Fluorescence Lifetime Imaging (FLIM), we tagged the c-terminus of GluN1 with monomeric GFP, as a FRET donor, and tagged the c-terminus of PSD95 with mCherry as a FRET acceptor
Since FRET between GluN1GFP and PSD95-mCherry was greatly increased by GluN2 subunits co-expression in Human embryonic kidney 293 (HEK293) cells (Figure 2E, Figure S1B in File S1), these results suggest that native GluN2 subunits in neurons assembled with GluN1-GFP to mediate the interaction with PSD95-mCherry
Summary
The dynamic molecular reorganization that takes place in dendritic spines is under increasing investigation, as it supports synaptic remodeling, a process required for memory formation. The cytoplasmic tails (ctails) of the NMDAR are central hubs where several signaling proteins, such as kinases and phosphatases, and anchoring proteins, including PSD95, converge [3] Some of these kinases, namely CaMKII and Src, phosphorylate GluN2 subunits or PSD95 during synaptic plasticity [4,5]. Namely CaMKII and Src, phosphorylate GluN2 subunits or PSD95 during synaptic plasticity [4,5] Another process that concerns active GluN2 subunits of the NMDAR is the intervention of calpain, which can cleave their c-tails, under the regulation of kinases and PSD95 [6,7,8,9]. The changing ratio of GluN2A/2B during post-natal development [10,11,12,13] likely impacts on the involvement of calpain and these kinases in NMDAR signaling [14]
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