Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca 2+ influx via NMDA receptors. Here, we show that Ca 2+ /calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca 2+ -induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca 2+ -induced dissociation of PSD-95 from the postsynaptic membrane.

Structural plasticity of dendritic spines is a key component of the refinement of synaptic connections during learning. Recent studies highlight a novel role for the NMDA receptor (NMDAR), independent of ion flow, in driving spine shrinkage and LTD. Yet little is known about the molecular mechanisms that link conformational changes in the NMDAR to changes in spine size and synaptic strength. Here, using two-photon glutamate uncaging to induce plasticity at individual dendritic spines on hippocampal CA1 neurons from mice and rats of both sexes, we demonstrate that p38 MAPK is generally required downstream of non-ionotropic NMDAR signaling to drive both spine shrinkage and LTD. In a series of pharmacological and molecular genetic experiments, we identify key components of the non-ionotropic NMDAR signaling pathway driving dendritic spine shrinkage, including the interaction between NOS1AP (nitric oxide synthase 1 adaptor protein) and neuronal nitric oxide synthase (nNOS), nNOS enzymatic activity, activation of MK2 (MAPK-activated protein kinase 2) and cofilin, and signaling through CaMKII. Our results represent a large step forward in delineating the molecular mechanisms of non-ionotropic NMDAR signaling that can drive shrinkage and elimination of dendritic spines during synaptic plasticity.

Despite the central role PSD-95 plays in anchoring postsynaptic AMPARs, how PSD-95 itself is tethered to postsynaptic sites is not well understood. Here we show that the F-actin binding protein α-actinin binds to the very N terminus of PSD-95. Knockdown (KD) of α-actinin phenocopies KD of PSD-95. Mutating lysine at position 10 or lysine at position 11 of PSD-95 to glutamate, or glutamate at position 53 or glutamate and aspartate at positions 213 and 217 of α-actinin, respectively, to lysine impairs, in parallel, PSD-95 binding to α-actinin and postsynaptic localization of PSD-95 and AMPARs. These experiments identify α-actinin as a critical PSD-95 anchor tethering the AMPAR-PSD-95 complex to postsynaptic sites.

SUMMARY Experience-dependent refinement of neuronal connections is critically important for brain development and learning. Here, we show that ion-flow-independent NMDA receptor (NMDAR) signaling is required for the long-term dendritic spine growth that is a vital component of brain circuit plasticity. We find that inhibition of p38 mitogen-activated protein kinase (p38 MAPK), which is downstream of non-ionotropic NMDAR signaling in long-term depression (LTD) and spine shrinkage, blocks long-term potentiation (LTP)-induced spine growth but not LTP. We hypothesize that non-ionotropic NMDAR signaling drives the cytoskeletal changes that support bidirectional spine structural plasticity. Indeed, we find that key signaling components downstream of non-ionotropic NMDAR function in LTD-induced spine shrinkage are also necessary for LTP-induced spine growth. Furthermore, NMDAR conformational signaling with coincident Ca 2+ influx is sufficient to drive CaMKII-dependent long-term spine growth, even when Ca 2+ is artificially driven through voltage-gated Ca 2+ channels. Our results support a model in which non-ionotropic NMDAR signaling gates the bidirectional spine structural changes vital for brain plasticity.

The formation and stabilization of new dendritic spines is a key component of the experience-dependent neural circuit plasticity that supports learning, but the molecular maturation of nascent spines remains largely unexplored. The PSD95-family of membrane-associated guanylate kinases (PSD-MAGUKs), most notably PSD95, has a demonstrated role in promoting spine stability. However, nascent spines contain low levels of PSD95, suggesting that other members of the PSD-MAGUK family might act to stabilize nascent spines in the early stages of spiny synapse formation. Here, we used GFP-fusion constructs to quantitatively define the molecular composition of new spines, focusing on the PSD-MAGUK family. We found that PSD95 levels in new spines were as low as those previously associated with rapid subsequent spine elimination, and new spines did not achieve mature levels of PSD95 until between 12 and 20 h following new spine identification. Surprisingly, we found that the PSD-MAGUKs PSD93, SAP97, and SAP102 were also substantially less enriched in new spines. However, they accumulated in new spines more quickly than PSD95: SAP102 enriched to mature levels within 3 h, SAP97 and PSD93 enriched gradually over the course of 6 h. Intriguingly, when we restricted our analysis to only those new spines that persisted, SAP97 was the only PSD-MAGUK already present at mature levels in persistent new spines when first identified. Our findings uncover a key structural difference between nascent and mature spines, and suggest a mechanism for the stabilization of nascent spines through the sequential arrival of PSD-MAGUKs.