Activity modifies the protein composition of the PSD. Synaptic scaffolding proteins are particularly well-suited for the “driver's” role in PSD remodeling since their change can influence the levels of multiple interacting proteins. GKAP is a central member of the axis of major scaffolds in the PSD, consisting of PSD-95-GKAP-Shank10, 37
. However, compared to PSD-95 and Shank, which play various roles in synaptic function including glutamate receptor trafficking, synapse formation, and spine morphogenesis37, 38
, little is known about the cell biology and function of GKAP. In this paper, we defined a critical role for GKAP in activity-dependent turnover of PSD-95 and Shank in the PSDs and in homeostatic synaptic scaling.
UPS-dependent protein degradation has emerged as an important theme underlying synaptic plasticity4, 16, 39, 40
. A distinguished characteristic of activity-dependent turnover of PSD proteins is that groups of synaptic proteins are co-regulated, perhaps via control of master organizing proteins within the PSD4
. Several characteristics of GKAP fit well for an organizer role within the PSD. First, GKAP is an indispensable central linker for the assembly of PSD-95-GKAP-Shank complexes at synapses8
, and PSD-95, GKAP and Shank are mutually dependent on each other for stable accumulation at synapses12, 41
. Indeed, activity-dependent turnover of PSD-95 and Shank was dependent on the turnover of GKAP. Second, GKAP is one of the direct substrates of the UPS (see ; also Ref. 4
). Third, and most importantly, the synaptic accumulation of GKAP is controlled bi-directionally by synaptic activity. Interestingly, the level of activity is decoded by different Ca2+
influx through NMDA receptor triggers poly-ubiquitination and degradation of GKAP. On the other hand, Ca2+
influx through L-VDCC is required for the synaptic accumulation of GKAP during inactivity. However, it is unlikely that GKAP turnover controls all PSD protein remodeling processes. A group of PSD proteins showed opposite changes to GKAP in response to altered activity: e.g., NR2A and α-CaMKII4
. Furthermore, following GKAP turnover, synaptic scaling requires additional players like Plk2 to target other synaptic protein complexes for activity-dependent degradation by the UPS33, 36
The opposing functions of α and β-CaMKII in the bi-directional synaptic scaling have been well documented 22-24
but the specific regulatory targets of these enzymes have remained unclear. Our finding indicates that GKAP is the critical substrate of these CaMKII isoforms for the activity-dependent control of synaptic strength. How does the activation of α- versus β-CaMKII exert differential effects on GKAP turnover? Importantly, activity modulates synaptic levels of α- and β-CaMKII, rendering α-CaMKII as a dominant species during over-excitation, while leaving β-CaMKII as a major kinase during low-level activity21-24
. Therefore, we expect that Ca2+
influx through NMDA receptors during high activity preferentially acts through α-CaMKII, which translocates to the PSD during high activity, and recruits proteasomes to dendritic spines16
. On the other hand, Ca2+
influx through L-VDCCs during low level activity (activated by spontaneous release of glutamate and miniature EPSPs)42
preferentially stimulates β-CaMKII, which has higher sensitivity to Ca2+
, is associated with actin concentrated at the base of dendritic spine heads44
, where L-VDCCs protein are also localized45
What are the underlying molecular mechanisms for GKAP depletion and accumulation at synapses? Our results suggest a removal mechanism in which α-CaMKII phosphorylation in the N-terminal repeat region of GKAP disrupts GKAP interaction with PSD-95 and promotes UPS-dependent degradation (see Supplementary Fig. 9a
). While dissociation from PSD-95 requires phosphorylation of S54 and S201, S54 phosphorylation was sufficient for poly-ubiquitination of GKAP. Notably, S54D mutant did not show Bic-induced reduction. This is probably due to the overexpression-induced dominant–negative effect of inhibiting poly-ubiquitination of S54D itself by saturating specific E3 ubiquitin ligase. GKAP is likely transported away from synapses before degradation by proteasomes, since preventing proteasome activity did not protect synaptic GKAP but rather produced large aggregates of GKAP in the soma. However, this view is different from the previous report proposing in situ degradation of GKAP at synapses4
. In addition, the report showed a completely opposite activity-dependent regulation of PSD-954
. At present the bases of these discrepancies are unclear but differences in the density of hippocampal neurons and the concentration of proteasome inhibitors applied to culture might have contributed. It is also unclear from our data whether ubiquitination of GKAP occurs at or near synapses. Further studies are necessary to address these questions.
For the accumulation mechanism for GKAP, as depicted in Supplementary Fig. 9b
, we propose that β-CaMKII phosphorylation of S340 and S384 in the DLC-binding domain of GKAP promotes the dissociation of GKAP from MVa-DLC2 motor protein complexes that transport GKAP to the base of the PSDs, and then the “unloaded” GKAP incorporates into the PSDs. For this, β-CaMKII association with the actin cytoskeleton was critical, presumably because it provides spatially favored position to regulate the interaction. A similar regulatory role of CaMKII was shown for MVa-mediated transport of GluR1 to synapses46
and for Kif17-mediated transport of Mint1-NMDAR complex in spines47
It is remarkable that deletion of R1 in GKAP was sufficient to block the both-directional activity-dependent turnover of GKAP, which is different from S54A mutation that blocked only Bic-induced removal from synapses. One major difference between these two mutants is the CaMKII-dependent dissociability from PSD-95. The S54A mutant possesses S201 that allows α-CaMKII to prevent GKAP interaction with PSD-95 by phosphorylation, as suggested by the impaired PSD-95 interaction of S54&201D mutant (). In contrast, unlike WT GKAP, ΔR1 mutant retained ΔR1–PSD-95 co-clusters even in the presence of constitutively active α-CaMKII in COS cells (Supplementary Fig. 10
). Thus, it is likely that deletion of the R1 likely induces changes in the overall conformation of the GKAP repeat region, so that the additional α-CaMKII regulation site in the R5 (S201) is masked, rendering GKAP resistant to α-CaMKII regulation of its interaction with PSD-95. These results suggest that the dissociation of GKAP from PSD-95 is required for the accumulation/recruitment of GKAP. Further studies are required to clarify how deletion of R1 prevents GKAP accumulation by inactivity. Furthermore, the CaMKII isoform-specific phosphorylation of GKAP at differential sites needs to be tested by additional biochemical experiments.
Intriguingly, the RNAi-mediated knockdown of GKAP not only prevented synaptic scaling measured by changes in mEPSC amplitude () but, unlike GKAP turnover mutant ΔR1, also had an additional effect of reducing mEPSC frequency. Since mEPSC frequency is mostly determined by the number of synapses, the data suggest that GKAP RNAi led to the loss of synapses. Thus, GKAP is not only important for the activity-dependent remodeling of synapses and synaptic scaling but also an essential scaffolding protein of the PSDs for the maintenance of excitatory synapses.
Bic is a GABAA
receptor antagonist commonly used for inducing synaptic scaling, and indirectly enhances overall excitatory activity in neurons by reducing inhibitory inputs to excitatory neurons. Synaptic scaling is thought to occur in all synapses2
, since cumulative histogram show a shift of entire mEPSC amplitude distribution toward smaller values after Bic treatment (). Consistent with this idea, cumulative distribution of GKAP cluster intensities revealed a similar shift after Bic treatment (Supplementary Fig. 11c
), indicating that Bic reduced the amount of GKAP from most synapses (if not all). Therefore, Bic has a global effect and, unlike synapse-specific Hebbian-type plasticity, synaptic scaling affects all synapses.
GKAP was shown to be poly-ubiquitinated by TRIM3 ubiquitin ligase48
, which raises a possibility that TRIM3 may involves in the CaMKII-mediated GKAP degradation. However, we were not able to observe a specific association of TRMI3 with pS54 peptide or S54D mutant by pull-down assays (data not shown), indicating that TRIM is unlikely involved in the CaMKII-dependent degradation of GKAP described here. CDK5 is another protein kinase involved in the GKAP degradation induced by soluble amyloid β49
. However, this also likely represents an independent GKAP regulatory mechanism, since the CDK5 phosphorylation sites in GKAP are different from the α-CaMKII sites, and more importantly, CaMKII inhibitors did not prevent GKAP degradation by soluble amyloid β49
Finally, recent studies on knockout mice lacking SAPAP-3, a GKAP family member highly expressed in the striatum, showed unexpected behavioral abnormalities similar to obsessive-compulsive disorder50
. Our findings on the critical role of GKAP/SAPAP in synapse remodeling and homeostatic plasticity offer potentially new ways to think about the pathophysiology of this condition.