CaMKII autophosphorylation is the only enzymatic event required for synaptic memory.

Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) plays a critical role in long-term potentiation (LTP), a well-established model for learning and memory through the enhancement of synaptic transmission. Biochemical studies indicate that CaMKII catalyzes a phosphotransferase (kinase) reaction of both itself (autophosphorylation) and of multiple downstream target proteins. However, whether either type of phosphorylation plays any role in the synaptic enhancing action of CaMKII remains hotly contested. We have designed a series of experiments to define the minimal requirements for the synaptic enhancement by CaMKII. We find that autophosphorylation of T286 and further binding of CaMKII to the GluN2B subunit are required both for initiating LTP and for its maintenance (synaptic memory). Once bound to the NMDA receptor, the synaptic action of CaMKII occurs in the absence of target protein phosphorylation. Thus, autophosphorylation and binding to the GluN2B subunit are the only two requirements for CaMKII in synaptic memory.

CaMKII autophosphorylation but not downstream kinase activity is required for synaptic memory.

CaMKII plays a critical role in long-term potentiation (LTP), a well-established model for learning and memory through the enhancement of synaptic transmission. Biochemical studies indicate that CaMKII catalyzes a phosphotransferase (kinase) reaction of both itself (autophosphorylation) and of multiple downstream target proteins. However, whether either type of phosphorylation plays any role in the synaptic enhancing action of CaMKII remains hotly contested. We have designed a series of experiments to define the minimal requirements for the synaptic enhancement by CaMKII. We find that autophosphorylation of T286 and further binding of CaMKII to the GluN2B subunit are required both for initiating LTP and for its maintenance (synaptic memory). Once bound to the NMDA receptor, the synaptic action of CaMKII occurs in the absence of kinase activity. Thus, autophosphorylation, together with binding to the GluN2B subunit, are the only two requirements for CaMKII in synaptic memory.

Contrastsing synaptic roles of MDGA1 and MDGA2.

Neurodevelopmental disorders are frequently linked to mutations in synaptic organizing molecules. MAM domain containing glycosylphosphatidylinositol anchor 1 and 2 (MDGA1 and MDGA2) are a family of synaptic organizers suggested to play an unusual role as synaptic repressors, but studies offer conflicting evidence for their localization. Using epitope-tagged MDGA1 and MDGA2 knock-in mice, we found that native MDGAs are expressed throughout the brain, peaking early in postnatal development. Surprisingly, endogenous MDGA1 was enriched at excitatory, but not inhibitory, synapses. Both shRNA knockdown and CRISPR/Cas9 knockout of MDGA1 resulted in cell-autonomous, specific impairment of AMPA receptor-mediated synaptic transmission, without affecting GABAergic transmission. Conversely, MDGA2 knockdown/knockout selectively depressed NMDA receptor-mediated transmission but enhanced inhibitory transmission. Our results establish that MDGA2 acts as a synaptic repressor, but only at inhibitory synapses, whereas both MDGAs are required for excitatory transmission. This nonoverlapping division of labor between two highly conserved synaptic proteins is unprecedented.

Synaptic memory and CaMKII.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) and long-term potentiation (LTP) were discovered within a decade of each other and have been inextricably intertwined ever since. However, like many marriages, it has had its up and downs. Based on the unique biochemical properties of CaMKII, it was proposed as a memory molecule before any physiological linkage was made to LTP. However, as reviewed here, the convincing linkage of CaMKII to synaptic physiology and behavior took many decades. New technologies were critical in this journey, including in vitro brain slices, mouse genetics, single-cell molecular genetics, pharmacological reagents, protein structure, and two-photon microscopy, as were new investigators attracted by the exciting challenge. This review tracks this journey and assesses the state of this marriage 40 years on. The collective literature impels us to propose a relatively simple model for synaptic memory involving the following steps that drive the process: 1) Ca2+ entry through N-methyl-d-aspartate (NMDA) receptors activates CaMKII. 2) CaMKII undergoes autophosphorylation resulting in constitutive, Ca2+-independent activity and exposure of a binding site for the NMDA receptor subunit GluN2B. 3) Active CaMKII translocates to the postsynaptic density (PSD) and binds to the cytoplasmic C-tail of GluN2B. 4) The CaMKII-GluN2B complex initiates a structural rearrangement of the PSD that may involve liquid-liquid phase separation. 5) This rearrangement involves the PSD-95 scaffolding protein, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), and their transmembrane AMPAR-regulatory protein (TARP) auxiliary subunits, resulting in an accumulation of AMPARs in the PSD that underlies synaptic potentiation. 6) The stability of the modified PSD is maintained by the stability of the CaMKII-GluN2B complex. 7) By a process of subunit exchange or interholoenzyme phosphorylation CaMKII maintains synaptic potentiation in the face of CaMKII protein turnover. There are many other important proteins that participate in enlargement of the synaptic spine or modulation of the steps that drive and maintain the potentiation. In this review we critically discuss the data underlying each of the steps. As will become clear, some of these steps are more firmly grounded than others, and we provide suggestions as to how the evidence supporting these steps can be strengthened or, based on the new data, be replaced. Although the journey has been a long one, the prospect of having a detailed cellular and molecular understanding of learning and memory is at hand.

Differential roles of CaMKII isoforms in phase separation with NMDA receptors and in synaptic plasticity.

Calcium calmodulin-dependent kinase II (CaMKII) is critical for synaptic transmission and plasticity. Two major isoforms of CaMKII, CaMKIIα and CaMKIIß, play distinct roles in synaptic transmission and long-term potentiation (LTP) with unknown mechanisms. Here, we show that the length of the unstructured linker between the kinase domain and the oligomerizing hub determines the ability of CaMKII to rescue the basal synaptic transmission and LTP defects caused by removal of both CaMKIIα and CaMKIIß (double knockout [DKO]). Remarkably, although CaMKIIß binds to GluN2B with a comparable affinity as CaMKIIα does, only CaMKIIα with the short linker forms robust dense clusters with GluN2B via phase separation. Lengthening the linker of CaMKIIα with unstructured "Gly-Gly-Ser" repeats impairs its phase separation with GluN2B, and the mutant enzyme cannot rescue the basal synaptic transmission and LTP defects of DKO mice. Our results suggest that the phase separation capacity of CaMKII with GluN2B is critical for its cellular functions in the brain.

Long-term potentiation reconstituted with an artificial TARP/PSD-95 complex.

The critical role of AMPA receptor (AMPAR) trafficking in long-term potentiation (LTP) of excitatory synaptic transmission is now well established, but the underlying molecular mechanism is still uncertain. Recent research suggests that PSD-95 captures AMPARs via an interaction with the AMPAR auxiliary subunits-transmembrane AMPAR regulatory proteins (TARPs). To determine if such interaction is a core minimal component of the AMPAR trafficking and LTP mechanism, we engineered artificial binding partners, which individually were biochemically and functionally dead but which, when expressed together, rescue binding and both basal synaptic transmission and LTP. These findings establish the TARP/PSD-95 complex as an essential interaction underlying AMPAR trafficking and LTP.

Synaptic memory survives molecular turnover.

Activation of Ca2+/calmodulin-dependent kinase II (CaMKII) plays a critical role in long-term potentiation (LTP), a long accepted cellular model for learning and memory. However, how LTP and memories survive the turnover of synaptic proteins, particularly CaMKII, remains a mystery. Here, we take advantage of the finding that constitutive Ca2+-independent CaMKII activity, acquired prior to slice preparation, provides a lasting memory trace at synapses. In slice culture, this persistent CaMKII activity, in the absence of Ca2+ stimulation, remains stable over a 2-wk period, well beyond the turnover of CaMKII protein. We propose that the nascent CaMKII protein present at 2 wk acquired its activity from preexisting active CaMKII molecules, which transferred their activity to newly synthesized CaMKII molecules and thus maintain the memory in the face of protein turnover.

Synaptic memory requires CaMKII.

Long-term potentiation (LTP) is arguably the most compelling cellular model for learning and memory. While the mechanisms underlying the induction of LTP ('learning') are well understood, the maintenance of LTP ('memory') has remained contentious over the last 20 years. Here, we find that Ca2+-calmodulin-dependent kinase II (CaMKII) contributes to synaptic transmission and is required LTP maintenance. Acute inhibition of CaMKII erases LTP and transient inhibition of CaMKII enhances subsequent LTP. These findings strongly support the role of CaMKII as a molecular storage device.

How the brain stores information is a question that has fascinated neuroscientists for well over a century. Two general ideas have emerged. The first is that groups of neurons hold information by staying active. The second is that they hold information by strengthening their connections to one another, making it easier for them to work together in the future. Scientists call this second idea 'long-term potentiation'. One of the molecules involved in long-term potentiation is a protein called calcium-calmodulin-dependent kinase II, or CaMKII for short. Blocking CaMKII, or deleting its gene, stops the connections between neurons from becoming stronger. This suggests neurons need CaMKII to learn, but it remains unclear whether neurons also use CaMKII to maintain neuronal memories after they have been created. If CaMKII does play a role in maintaining memories, blocking it after learning should reverse the learning process, but so far, experiments have not been able to show this. Tao et al. revisited these experiments to find out more. They examined slices of brain tissue from mice that had been treated with fast-acting CaMKII inhibitors. It took tens of minutes, but the inhibitors were able to reverse long-term potentiation, both for newly acquired neuronal memories and for older memories that had formed when the mice were alive. The choice of CaMKII inhibitor and the time lag could explain why scientists have not observed the effect before. Understanding long-term potentiation is a fundamental part of understanding learning and memory. It could also reveal more about the opposite phenomenon: long-term depression. This is a type of learning where the connections between neurons become weaker. Long-term depression also takes tens of minutes to occur, suggesting that future research into CaMKII might shed light on how it works.

AMPA receptor trafficking and LTP: Carboxy-termini, amino-termini and TARPs.

AMPA receptors (AMPARs) are fundamental elements in excitatory synaptic transmission and synaptic plasticity in the CNS. Long term potentiation (LTP), a form of synaptic plasticity which contributes to learning and memory formation, relies on the accumulation of AMPARs at the postsynapse. This phenomenon requires the coordinated recruitment of different elements in the AMPAR complex. Based on recent research reviewed herein, we propose an updated AMPAR trafficking and LTP model which incorporates both extracellular as well as intracellular mechanisms. This article is part of the special Issue on 'Glutamate Receptors - AMPA receptors'.

MAGUKs are essential, but redundant, in long-term potentiation.

This study presents evidence that the MAGUK family of synaptic scaffolding proteins plays an essential, but redundant, role in long-term potentiation (LTP). The action of PSD-95, but not that of SAP102, requires the binding to the transsynaptic adhesion protein ADAM22, which is required for nanocolumn stabilization. Based on these and previous results, we propose a two-step process in the recruitment of AMPARs during LTP. First, AMPARs, via TARPs, bind to exposed PSD-95 in the PSD. This alone is not adequate to enhance synaptic transmission. Second, the AMPAR/TARP/PSD-95 complex is stabilized in the nanocolumn by binding to ADAM22. A second, ADAM22-independent pathway is proposed for SAP102.

LGI1-ADAM22-MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention.

Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1-ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv1 channels, and LRRTM4-Neurexin adhesion molecules. Adam22ΔC5/ΔC5 knock-in mice devoid of the ADAM22-MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1-ADAM22-MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

Long-term potentiation is independent of the C-tail of the GluA1 AMPA receptor subunit.

We tested the proposal that the C-terminal domain (CTD) of the AMPAR subunit GluA1 is required for LTP. We found that a knock-in mouse lacking the CTD of GluA1 expresses normal LTP and spatial memory, assayed by the Morris water maze. Our results support a model in which LTP generates synaptic slots, which capture passively diffusing AMPARs.

Phase Separation-Mediated TARP/MAGUK Complex Condensation and AMPA Receptor Synaptic Transmission.

Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARPs) modulate AMPAR synaptic trafficking and transmission via disc-large (DLG) subfamily of membrane-associated guanylate kinases (MAGUKs). Despite extensive studies, the molecular mechanism governing specific TARP/MAGUK interaction remains elusive. Using stargazin and PSD-95 as the representatives, we discover that the entire tail of stargazin (Stg_CT) is required for binding to PSD-95. The PDZ binding motif (PBM) and an Arg-rich motif upstream of PBM conserved in TARPs bind to multiple sites on PSD-95, thus resulting in a highly specific and multivalent stargazin/PSD-95 complex. Stargazin in complex with PSD-95 or PSD-95-assembled postsynaptic complexes form highly concentrated and dynamic condensates via phase separation, reminiscent of stargazin/PSD-95-mediated AMPAR synaptic clustering and trapping. Importantly, charge neutralization mutations in TARP_CT Arg-rich motif weakened TARP's condensation with PSD-95 and impaired TARP-mediated AMPAR synaptic transmission in mice hippocampal neurons. The TARP_CT/PSD-95 interaction mode may have implications for understanding clustering of other synaptic transmembrane proteins.

SynGO: An Evidence-Based, Expert-Curated Knowledge Base for the Synapse.

Synapses are fundamental information-processing units of the brain, and synaptic dysregulation is central to many brain disorders ("synaptopathies"). However, systematic annotation of synaptic genes and ontology of synaptic processes are currently lacking. We established SynGO, an interactive knowledge base that accumulates available research about synapse biology using Gene Ontology (GO) annotations to novel ontology terms: 87 synaptic locations and 179 synaptic processes. SynGO annotations are exclusively based on published, expert-curated evidence. Using 2,922 annotations for 1,112 genes, we show that synaptic genes are exceptionally well conserved and less tolerant to mutations than other genes. Many SynGO terms are significantly overrepresented among gene variations associated with intelligence, educational attainment, ADHD, autism, and bipolar disorder and among de novo variants associated with neurodevelopmental disorders, including schizophrenia. SynGO is a public, universal reference for synapse research and an online analysis platform for interpretation of large-scale -omics data (https://syngoportal.org and http://geneontology.org).

The STEP61 interactome reveals subunit-specific AMPA receptor binding and synaptic regulation.

Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific protein phosphatase that regulates a variety of synaptic proteins, including NMDA receptors (NAMDRs). To better understand STEP's effect on other receptors, we used mass spectrometry to identify the STEP61 interactome. We identified a number of known interactors, but also ones including the GluA2 subunit of AMPA receptors (AMPARs). We show that STEP61 binds to the C termini of GluA2 and GluA3 as well as endogenous AMPARs in hippocampus. The synaptic expression of GluA2 and GluA3 is increased in STEP-KO mouse brain, and STEP knockdown in hippocampal slices increases AMPAR-mediated synaptic currents. Interestingly, STEP61 overexpression reduces the synaptic expression and synaptic currents of both AMPARs and NMDARs. Furthermore, STEP61 regulation of synaptic AMPARs is mediated by lysosomal degradation. Thus, we report a comprehensive list of STEP61 binding partners, including AMPARs, and reveal a central role for STEP61 in differentially organizing synaptic AMPARs and NMDARs.

Mechanisms underlying the synaptic trafficking of the glutamate delta receptor GluD1.

Ionotropic glutamate delta receptors do not bind glutamate and do not generate ionic current, resulting in difficulty in studying the function and trafficking of these receptors. Here, we utilize chimeric constructs, in which the ligand-binding domain of GluD1 is replaced by that of GluK1, to examine its synaptic trafficking and plasticity. GluD1 trafficked to the synapse, but was incapable of expressing long-term potentiation (LTP). The C-terminal domain (CT) of GluD1 has a classic PDZ-binding motif, which is critical for the synaptic trafficking of other glutamate receptors, but we found that its binding to PSD-95 was very weak, and deleting the PDZ-binding motif failed to alter synaptic trafficking. However, deletion of the entire CT abolished synaptic trafficking, but not surface expression. We found that mutation of threonine (T) T923 to an alanine disrupted synaptic trafficking. Therefore, GluD1 receptors have strikingly different trafficking mechanisms compared with AMPARs. These results highlight the diversity of ionotropic glutamate receptor trafficking rules at a single type of synapse. Since this receptor is genetically associated with schizophrenia, our findings may provide an important clue to understand schizophrenia.

Isoform-specific cleavage of neuroligin-3 reduces synapse strength.

The assembly and maintenance of synapses are dynamic processes that require bidirectional contacts between the pre- and postsynaptic structures. A network of adhesion molecules mediate this physical interaction between neurons. How synapses are disassembled and if there are distinct mechanisms that govern the removal of specific adhesion molecules remain unclear. Here, we report isoform-specific proteolytic cleavage of neuroligin-3 in response to synaptic activity and protein kinase C signaling resulting in reduced synapse strength. Although neuroligin-1 and neuroligin-2 are not directly cleaved by this pathway, when heterodimerized with neuroligin-3, they too undergo proteolytic cleavage. Thus protein kinase C-dependent cleavage is mediated through neuroligin-3. Recent studies on glioma implicate the neuroligin-3 ectodomain as a mitogen. Here we demonstrate: (1) there are mechanisms governing specific adhesion molecule remodeling; (2) neuroligin-3 is a key regulator of neuroligin cleavage events; and (3) there are two cleavage pathways; basal and activity-dependent that produce the mitogenic form of neuroligin-3.

Author Correction: The CaMKII/NMDA receptor complex controls hippocampal synaptic transmission by kinase-dependent and independent mechanisms.

The originally published version of this Article contained errors in Figure 5, for which we apologise. In panel c, the scatter graph was inadvertently replaced with a scatter graph comprising a subset of data points from panel d. Furthermore, the legends to Figures 5c and 5d were inverted. These errors have now been corrected in both the PDF and HTML versions of the Article, and the incorrect version of Fig. 5c is presented in the Author Correction associated with this Article.

Signal peptide represses GluK1 surface and synaptic trafficking through binding to amino-terminal domain.

Kainate-type glutamate receptors play critical roles in excitatory synaptic transmission and synaptic plasticity in the brain. GluK1 and GluK2 possess fundamentally different capabilities in surface trafficking as well as synaptic targeting in hippocampal CA1 neurons. Here we find that the excitatory postsynaptic currents (EPSCs) are significantly increased by the chimeric GluK1(SPGluK2) receptor, in which the signal peptide of GluK1 is replaced with that of GluK2. Coexpression of GluK1 signal peptide completely suppresses the gain in trafficking ability of GluK1(SPGluK2), indicating that the signal peptide represses receptor trafficking in a trans manner. Furthermore, we demonstrate that the signal peptide directly interacts with the amino-terminal domain (ATD) to inhibit the synaptic and surface expression of GluK1. Thus, we have uncovered a trafficking mechanism for kainate receptors and propose that the cleaved signal peptide behaves as a ligand of GluK1, through binding with the ATD, to repress forward trafficking of the receptor.