Synaptic adhesion molecules regulate multiple steps of synapse formation and maturation. The great diversity of neuronal synapses predicts the presence of a large number of adhesion molecules that control synapse formation through trans-synaptic and heterophilic adhesion. We identified a previously unknown trans-synaptic interaction between netrin-G ligand-3 (NGL-3), a postsynaptic density (PSD) 95-interacting postsynaptic adhesion molecule, and leukocyte common antigen-related (LAR), a receptor protein tyrosine phosphatase. NGL-3 and LAR expressed in heterologous cells induced pre- and postsynaptic differentiation in contacting axons and dendrites of cocultured rat hippocampal neurons, respectively. Neuronal overexpression of NGL-3 increased presynaptic contacts on dendrites of transfected neurons. Direct aggregation of NGL-3 on dendrites induced coclustering of excitatory postsynaptic proteins. Knockdown of NGL-3 reduced the number and function of excitatory synapses. Competitive inhibition by soluble LAR reduced NGL-3-induced presynaptic differentiation. These results suggest that the trans-synaptic adhesion between NGL-3 and LAR regulates excitatory synapse formation in a bidirectional manner.

Mitogen-activated protein kinases (MAPKs) control neuronal synaptic function; however, little is known about the synaptic substrates regulated by MAPKs. A phosphopeptide library incorporating the MAPK consensus motif (PX(pS/pT)P where pS is phosphoserine and pT is phosphothreonine) was used to raise a phosphospecific antibody that detected MAPK-mediated phosphorylation. The antibody (termed "5557") recognized a variety of phosphoproteins in the brain, many of which were enriched in postsynaptic density fractions. The immunoblot pattern changed rapidly in response to altered synaptic activity and with the inhibition of specific MAPKs and protein phosphatases. By immunoaffinity purification with 5557 antibody followed by mass spectrometry, we identified 449 putative MAPK substrates of which many appeared dynamically regulated in neuron cultures. Several of the novel candidate MAPK substrates were validated by in vitro phosphorylation assays. Additionally 82 specific phosphorylation sites were identified in 34 proteins, including Ser-447 in delta-catenin, a component of the cadherin adhesion complex. We further raised another phosphospecific antibody to confirm that delta-catenin Ser-447 is modified in neurons by the MAPK JNK in a synaptic activity-dependent manner. Ser-447 phosphorylation by JNK appears to be correlated with delta-catenin degradation, and a delta-catenin mutant defective in Ser-447 phosphorylation showed enhanced ability to promote dendrite branching in cultured neurons. Thus, phosphomotif-based affinity purification is a powerful approach to identify novel substrates of MAPKs in vivo and to reveal functionally significant phosphorylation events.

BACKGROUND: Long-term depression (LTD) in the hippocampus can be induced by activation of different types of G-protein coupled receptors, in particular metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors (mAChRs). Since mGluRs and mAChRs activate the same G-proteins and isoforms of phospholipase C (PLC), it would be expected that these two forms of LTD utilise the same molecular mechanisms. However, we find a distinct mechanism of LTD involving GRIP and liprin-alpha.

RESULTS: Whilst both forms of LTD require activation of tyrosine phosphatases and involve internalisation of AMPARs, they use different molecular interactions. Specifically, mAChR-LTD, but not mGluR-LTD, is blocked by peptides that inhibit the binding of GRIP to the AMPA receptor subunit GluA2 and the binding of GRIP to liprin-alpha. Thus, different receptors that utilise the same G-proteins can regulate AMPAR trafficking and synaptic efficacy via distinct molecular mechanisms.

CONCLUSION: Our results suggest that mAChR-LTD selectively involves interactions between GRIP and liprin-alpha. These data indicate a novel mechanism of synaptic plasticity in which activation of M1 receptors results in AMPAR endocytosis, via a mechanism involving interactions between GluA2, GRIP and liprin-alpha.

Long-term depression (LTD) is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR)-dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs) via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD-95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner.

Experience-dependent changes in the structure of dendritic spines may contribute to learning and memory. Encoded by three genes, the Shank family of postsynaptic scaffold proteins are abundant and enriched in the postsynaptic density (PSD) of central excitatory synapses. When expressed in cultured hippocampal neurons, Shank promotes the maturation and enlargement of dendritic spines. Recently, Shank3 has been genetically implicated in human autism, suggesting an important role for Shank proteins in normal cognitive development. Here, we report the phenotype of Shank1 knock-out mice. Shank1 mutants showed altered PSD protein composition; reduced size of dendritic spines; smaller, thinner PSDs; and weaker basal synaptic transmission. Standard measures of synaptic plasticity were normal. Behaviorally, they had increased anxiety-related behavior and impaired contextual fear memory. Remarkably, Shank1-deficient mice displayed enhanced performance in a spatial learning task; however, their long-term memory retention in this task was impaired. These results affirm the importance of Shank1 for synapse structure and function in vivo, and they highlight a differential role for Shank1 in specific cognitive processes, a feature that may be relevant to human autism spectrum disorders.

Homeostatic plasticity keeps neuronal spiking output within an optimal range in the face of chronically altered levels of network activity. Little is known about the underlying molecular mechanisms, particularly in response to elevated activity. We report that, in hippocampal neurons experiencing heightened activity, the activity-inducible protein kinase Polo-like kinase 2 (Plk2, also known as SNK) was required for synaptic scaling-a principal mechanism underlying homeostatic plasticity. Synaptic scaling also required CDK5, which acted as a "priming" kinase for the phospho-dependent binding of Plk2 to its substrate SPAR, a postsynaptic RapGAP and scaffolding molecule that is degraded following phosphorylation by Plk2. RNAi knockdown of SPAR weakened synapses, and overexpression of a SPAR mutant resistant to Plk2-dependent degradation prevented synaptic scaling. Thus, priming phosphorylation of the Plk2 binding site in SPAR by CDK5, followed by Plk2 recruitment and SPAR phosphorylation-degradation, constitutes a molecular pathway for neuronal homeostatic plasticity during chronically elevated activity.

Homeostatic plasticity mechanisms stabilize the activity of a neuron or neuronal circuit during prolonged periods of increased network activity and have been proposed to function in the prevention of epilepsy. How homeostatic plasticity is achieved at the molecular level during hyperactivity states in general, and during epileptiform activity in particular, is unclear. Using organotypic hippocampal slice cultures as a model system, we found that the protein kinase Polo-like kinase 2 (Plk2) was induced during prolonged epileptiform activity and was required for the activity-dependent reduction in membrane excitability of pyramidal neurons. Disruption of Plk2 function by dominant-negative or RNA interference not only blocked the downregulation of membrane excitability during epileptiform activity, but also unmasked a slow and progressive potentiation in synaptic strength that prevented the ability of the slice to undergo long-term potentiation. Thus, Plk2 function is required to prevent escalating potentiation and maintain synapses in a plastic state during epileptiform activity in hippocampal slice cultures.

Within the Ras superfamily of GTPases, Rap1 and Rap2 are the closest homologs to Ras. In non-neural cells, Rap signaling can antagonize Ras signaling. In neurons, Rap also seems to oppose Ras in terms of synaptic function. Whereas Ras is critical for long-term potentiation (LTP), Rap1 has been shown to be required for long-term depression (LTD), and Rap2 has been implicated in depotentiation. Moreover, active Rap1 and Rap2 cause loss of surface AMPA receptors and reduced miniature EPSC amplitude and frequency in cultured neurons. The role of Rap signaling in vivo, however, remains poorly understood. To study the function of Rap2 in the brain and in behavior, we created transgenic mice expressing either constitutively active (Rap2V12) or dominant-negative (Rap2N17) mutants of Rap2 in postnatal forebrain. Multiple lines of Rap2N17 mice showed only weak expression of the transgenic protein, and no phenotype was observed. Rap2V12 mice displayed fewer and shorter dendritic spines in CA1 hippocampal neurons, and enhanced LTD at CA3-CA1 synapses. Behaviorally, Rap2V12 mice showed impaired spatial learning and defective extinction of contextual fear, which correlated with reduced basal phosphorylation of extracellular signal-regulated kinase (ERK) and blunted activation of ERK during fear extinction training. Our data support the idea that Rap2 opposes Ras-ERK signaling in the brain, thereby inhibiting dendritic spine development/maintenance, promoting synaptic depression rather than LTP, and impairing learning. The findings also implicate Rap2 signaling in fear extinction mechanisms, which are thought to be aberrant in anxiety disorders and posttraumatic stress disorder.